Treatment of hematologic disorders

ABSTRACT

The inventors have discovered that hematologic disorders, e.g., both neoplastic (hematologic cancers) and non-neoplastic conditions, can be treated by the induction of mixed chimerism using myeloreductive, but not myeloablative, conditioning. Methods of the invention reduce GVHD, especially GVHD associated with mismatched allogeneic or xenogeneic donor tissue, yet provide, for example, significant graft-versus-leukemia (GVL) effect and the like.

GOVERNMENT FUNDING

[0001] The work herein was supported by a grant from the NationalInstitutes of Health. The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0002] The invention relates to the treatment of hematologic disorders,e.g., disorders characterized by unwanted cells of hematopoietic origin,e.g., hematologic cancers.

[0003] Bone marrow transplantation (BMT) has yet to realize its fullpotential for the treatment of hematologic malignancies. A majorobstacle to further advancement is graft-versus-host disease (GVHD),which has been prevented by removing T cells from the donor marrow.Unfortunately, T cell depletion has been associated with increased ratesof engraftment failure and leukemic relapse. Despite improvements inpharmacologic GVHD prophylaxis, severe acute and chronic GVHD are stillmajor complications of HLA-matched sibling bone marrow transplantation.Immunosuppressive drugs used for GVHD prophylaxis may also increase therelapse rate for certain types of leukemia. The patients receivingallogeneic BMT are, nevertheless, a fortunate select group: mostpatients do not have an HLA-matched sibling or a phenotypically matchedunrelated donor, and therefore do not have the option of BMT. Attemptsto perform BMT between strongly HLA-mismatched donor-recipient pairshave been associated with a prohibitively high incidence of severe GVHDand of failure of engraftment. Furthermore, a large fraction ofleukemias and lymphomas afflict older patients who are more prone to thedevelopment of GVHD than are younger persons, and who therefore are notgenerally considered candidates for BMT, despite the lack of othercurative options.

SUMMARY OF THE INVENTION

[0004] The inventors have discovered that hematologic disorders, e.g.,both neoplastic (hematologic cancers) and non-neoplastic conditions, canbe treated by the induction of mixed chimerism in the absence of wholebody irradiation (total myeloablation protocols) or other myeloablativetreatment. Methods of the invention reduce GVHD, especially GVHDassociated with mismatched allogeneic or xenogeneic donor tissue, yetprovide significant graft-versus-leukemia (GVL) effect and the like.

[0005] Certain embodiments of the subject methods also featurepreparative regimens which minimize or eliminate the need formyeloablative treatment, e.g., hematopoietic space-creating irradiation,especially, preparative whole body irradiation.

[0006] One aspect of the present invention provides a method fortreating a subject having a hematologic disorder comprising: (i)administering a myeloreductive nonmyeloablative treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, and (ii) introducing into the subject,allogeneic donor hematopoietic stem cells (donor stem cells) to formchimeric bone marrow in the subject.

[0007] In certain embodiments, the myeloreductive treatment includestreating the subject with an immunosuppressant regimen, prior tointroduction of the donor stem cells, in an amount sufficient to preventrejection of the donor stem cells.

[0008] Likewise, the method can include a further step of treating thesubject with an immunosuppressant regimen, after introduction of thedonor stem cells, in an amount sufficient to prevent a graft-versus-hostresponse mediated by the donor stem cells.

[0009] Such immunosuppressant regimens can include, independently forpre- and post-transplantation is both are carried out, a treatment ofthe subject which inactivates and/or depletes host T-lymphocytes and/ornatural killer (NK) cells in the subject. For example, theimmunosuppressant regimen includes treatment with T cell-depletinganti-CD4 and/or CD8 antibodies, such as anti-thymocyte globulin (ATG),OKT3 (Orthoclone OKT3 monoclonal antibody, Ortho Pharmaceutical Corp),LO-CD2a (U.S. Pat. No. 5,730,979), or Minnesota anti-lymphoblastglobulin (MALG). Preferably, the immunosuppressant regimen, both beforeand after transplantation, includes administration of ATG.

[0010] Moreover, the immunosuppressant regimen can include treatmentwith thymic irradiation. Preferably, the pre-transplantationimmunosuppressant conditioning includes administration of ATG and thymicirradiation.

[0011] In other embodiments, the immunosuppressant regimen includestreatment with one or more of a macrolide immunosuppressant,azathioprine, steroids (e.g., prednisone, methyl prednisolone),sub-lethal nonmyeloablative irradiation of lymphocyte-containing tissue,or costimulatory blocking agents (e.g., anti-CD40 ligands, CTLA4Igfusion proteins, see, e.g., Lenschow et al., (1992) Science 257:789; andTurka et al., (1992) PNAS 89:11102).

[0012] In certain embodiments, the myeloreductive treatment includestreating the subject, prior to introduction of the donor stem cells,with an cytoreductive agent selected from one or more of alkylatingagents (e.g., nitrogen mustards [such as mechloretamine],cyclophosphamide, melphalan and chlorambucil), alkyl sulphonates (e.g.,busulphan), nitrosoureas (e.g., carmustine, lomustine, semustine andstreptozocine), triazenes (e.g., dacarbazine), antimetabolites (e.g.,folic acid analogs such as methotrexate), pyrimidine analogs (e.g.fluorouracil and cytarabine), purine analogs (e.g., fludarabine,idarubicin, cytosine arabinoside, mercaptopurine and thioguanine), vincaalkaloids (e.g., vinblastine, vincristine and vendesine),epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g.,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin andmitomycin), dibromomannitol, deoxyspergualine, dimethyl myleran andthiotepa.

[0013] Preferably, the myeloreductive treatment includes treating thesubject with cyclophosphamide.

[0014] Preferably, the pre-transplantation conditioning includesadministration of ATG and cyclophosphamide, and thymic irradiation.Preferably the cyclophosphamide, or other cytoreductive agents, aresubstantially cleared from the patient so as not inhibit proliferationof the transplanted stem cells.

[0015] An important use of the subject method is for allogeneictransplantation of donor stem cells which are mismatched, with respectto the subject, at one or more HLA class II antigens.

[0016] Another important use of the subject method is for allogeneictransplantation of donor stem cells which are mismatched, with respectto the subject, at two or more HLA antigens (either HLA class I or II orboth).

[0017] In preferred embodiments, the donor stem cells are provided asallogeneic bone marrow, mobilized peripheral blood cells, or cord bloodcells.

[0018] The donor stem cells, in some instances, can be expanded ex vivofor transplantation.

[0019] In preferred embodiments, the donor stem cells are from the samespecies as the subject. However, the present application alsospecifically contemplates that the donor stem cells are xenogeneic stemcells from a different species than the subject. In xenogeneic methods,the subject is a mammal, preferably a primate and more preferably ahuman. The donor mammal can be, by way of example, a swine, e.g., aminiature swine, or a nonhuman primate. In xenogeneic methods the donorof stem cells and the donor of leukocytes need not be the sameindividual but can be from different individuals which are MHC matchedor highly inbred, e.g., inbred miniature swine which are MHC matched.

[0020] In preferred embodiments, the subject is a human, and even morepreferably, the subject is a human and donor stem cells are from anotherhuman.

[0021] The methods of the present invention can be used to treat a widerange of hematologic disorders, including neoplastic proliferation ofhematopoetic cells, such as lymphoblastic leukemia, myelogenousleukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma and myelodysplasticsyndrome. As described herein, the subject method can be used to treathematologic disorders which are refractory to chemotherapy, such achemorefactory Non-Hodgkin's lymphoma.

[0022] In other embodiments, the subject method can be used to treathematologic disorders which are non-malignant, such as erythrocyteabnormalities or immune system disorders. For example, the instantmethod can be used to treat hemoglobinopathies, e.g., sickle cellanemia, aplastic anemia or thalassemia. The subject method also can beused as part of a treatment regimen for autoimmune disorders as well asimmunodeficiencies.

[0023] In several embodiments, particularly where little, and preferablyno GVHD is detected post-transplantation (e.g., at least 14 days, andmore preferably at least 25, 30 or even 35 days), the subject methodincludes the further step of administering allogeneic donor leukocytesto the subject after introduction of the donor stem cells. Theadministration of donor leukocytes should be delayed sufficiently fromthe time of any hematopoietic space creating treatment such that thelevel of pro-inflammatory cytokines induced by the space creatingtreatment has subsided sufficiently to reduce or substantially eliminateGVHD from the donor leukocytes.

[0024] The subject method can also include the management of GVHDresponses post-transplantation by administration of immunosuppressants,or by use of engineered stem cells which give rise to small moleculeablatable T cells or other hematopoietic cells. See, for example, U.S.Pat. No. 5,834,266.

[0025] In another aspect, the invention features a method of treating anon-neoplastic disorder or a hemoglobinopathy, e.g., sickle cell anemia,aplastic anemia, thalassemia Thus, in one preferred embodiment, thesubject method comprises: (i) identifying a patient having a neoplastichematopoetic disorder, (ii) administering a myeloreductive treatment tothe subject in sufficient amount such that mixed hematopoietic chimerismcan be induced in the subject, and (iii) introducing into the subject,allogeneic donor hematopoietic stem cells (donor stem cells) to formstable mixed chimeric bone marrow in the subject.

[0026] In another preferred embodiment, the subject method comprises:(i) identifying a patient having a neoplastic hematopoetic disorder,(ii) administering a myeloreductive treatment to the subject insufficient amount such that mixed hematopoietic chimerism can be inducedin the subject, and (iii) introducing into the subject, allogeneic donorhematopoietic stem cells (donor stem cells) to form mixed chimeric bonemarrow in the subject, wherein the donor stem cells are mismatched, withrespect to the patient, at one or more class II HLA antigens.

[0027] In still another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a myeloreductive treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, and (iii) introducing into the subject,allogeneic donor hematopoietic stem cells (donor stem cells) to formmixed chimeric bone marrow in the subject, wherein the donor stem cellsare mismatched, with respect to the patient, at two or more HLAantigens, e.g., class I and/or class II.

[0028] In yet another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a myeloreductive treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, (iii) introducing into the subject, allogeneicdonor hematopoietic stem cells (donor stem cells) to form mixed chimericbone marrow in the subject, and (iv) administering apost-transplantation immunosuppression regimen for suppressing ordepleting T-cells in the transplanted donor stem cells.

[0029] In yet another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a pre-transplantation conditioning to thesubject in sufficient amount such that mixed hematopoietic chimerism canbe induced in the subject, which pre-transplantation conditioningincludes treating the cells with cyclophosphamide, ATG and thymicirradiation in an amount sufficient to reduce rejection of transplanteddonor stem cells; and (iii) introducing into the subject, allogeneicdonor hematopoietic stem cells (donor stem cells) to form mixed chimericbone marrow in the subject, and (iv) administering ATG to the subjectpost-transplant for suppressing or depleting T-cells in the transplanteddonor stem cells.

[0030] Another aspect of the present invention relates to the use ofdonor allogeneic stem cells in the manufacture of a medicament for thetreatment of a hematologic disorder, wherein the medicament administeredto a patient conditioned with myeloreductive non-myeloablativetreatment, and in an amount sufficient to form chimeric bone marrow inthe subject.

[0031] Still another aspect of the present invention provides a kit forallogeneic hematopoietic stem cell transplantation. The kit includescyclophosphamide in an amount sufficient to reduce rejection oftransplanted donor stem cells when administered to a patientpre-transplantation, and ATG in an amount sufficient to reduce rejectionof transplanted donor stem cells when administered to a patientpre-transplantation and suppress T-cells in transplanted donor stemcells. The kit may also include a labeled antibody for detectingleukocytes as part of a step of determining chimerism of a treatedanimal. The kit may also include HLA-mismatched donor stem cells, e.g.,allogeneic BMT, mobilized peripheral blood cells, cord blood cells, orhematopoietic cells derived from cultured stem/progenitor cells.

[0032] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

[0033] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1: Time course of mixed WBC chimerism in Patient 1. Thepercentage of donor (open bar) and host (solid bar) cells in each WBCpopulation is shown over time. Each WBC population is normalized to100%, so that the proportion of that particular population that is ofdonor vs. host origin is presented.

[0035]FIG. 2: Mixed chimerism in WBC of Patient 1 one year post-BMT.Lymphocyte, monocyte and granulocyte gates are shown on the forwardscatter (FSC)× side scatter (SSC) contour plot, and staining patternswith anti-HLA-A9 mAb are shown for lymphocytes (right top panel),monocytes (right middle panel) and granulocytes (right lower panel). Thedonor was HLA-A9 -negative, whereas the host was A9-positive. The barsabove the histograms denote the populations considered to beHLA-A9-negative.

DETAILED DESCRIPTION OF THE INVENTION

[0036] (i) Overview

[0037] Bone marrow transplantation (BMT) has previously been limitedfrom its full potential for the treatment of hematologic malignancies,due to the fact that most patients in need of an allogeneic BMT do nothave HLA-matched donors available. A major obstacle to furtheradvancement of HLA-mismatched donor BMT following standard myeloablativeconditioning therapy for hematologic malignancies has been theoccurrence of severe graft-versus-host disease (GVHD) and graft failure.

[0038] The present invention provides an approach which can be used inhuman patients in which lymphohematopoietic graft-versus-host (GVH)reactions, e.g., graft-versus-leukemia or graft-versus-lymphoma, canoccur without GVHD. The non-myeloablative conditioning of the subjectmethod permits the generation of mixed hematopoietic chimeras producedacross MHC barriers, including, significantly, class II mismatches. Themore potent alloresponses generated against MHC disparities compared tothose against minor histocompatibility antigens usually elicits severeGVHD, which has been the major impediment to HLA-mismatched BMT (Cliftet al. (1987) Ann Rev Immunol. 5:43-64. In certain HLA-mismatched BMTdescribed herein, the subject protocols could not completely suppressGVHD, but in many instances it was surprisingly mild and amenable tocorticosteroid therapy and the like.

[0039] (ii) Definitions

[0040] For convenience, certain terms employed in the specification,examples, and claims are collected here.

[0041] “Stromal tissue”, as used herein, refers to the supporting tissueor matrix of an organ, as distinguished from its functional elements orparenchyma.

[0042] “Hematopoietic space”, as used herein, refers to a conditioncreated in the bone marrow which promotes engraftment of administeredstem cells. In the art, hematopoietic space has often been created byirradiation of the bone marrow with whole body irradiation, but themethods of the invention generally use nonmyeloablative treatments.

[0043] “Hematopoietic stem cell”, as used herein, refers to a cell,e.g., a bone marrow cell, or a fetal liver or spleen cell, which iscapable of developing into all myeloid and lymphoid lineages and byvirtue of being able to self-renew can provide long term hematopoieticreconstitution. Purified preparations of hematopoietic cells orpreparations, such as bone marrow, which include other cell types, canbe used in methods of the invention. Although not wishing to be bound bytheory, it is believed that the hematopoietic stem cells home to a sitein the recipient. The preparation should include immature cells, i.e.,undifferentiated hematopoietic stem cells; these desired cells can beseparated out of a preparation or a complex preparation can beadministered. E.g., in the case of bone marrow stem cells, the desiredprimitive cells can be separated out of a preparation or a complex bonemarrow sample including such cells can be used. Hematopoietic stem cellscan be from fetal, neonatal, immature or mature animals. Stem cellsderived from the cord blood of the recipient or the donor can be used inmethods of the invention. See U.S. Pat. No. 5,192,553, herebyincorporated by reference, and U.S. Pat. No. 5,004,681, herebyincorporated by reference.

[0044] A “peripheral blood stem cell” is a cell with the potential toproduce all the components of blood that is obtained from peripheralblood rather than from bone marrow.

[0045] An “immunosuppressive agent”, as used herein, is an agent, e.g.,a chemical agent, e.g., a drug, which, when administered at anappropriate dosage, results in the inhibition of T cells. Examples ofsuch agents are cyclosporine, FK-506, and rapamycin.

[0046] “Thymic or lymph node or thymocytes or T cell”, as used herein,refers to thymocytes or T cells which are resistant to inactivation bytraditional methods of T cell inactivation, e.g., inactivation by asingle intravenous administration of anti-T cell antibodies, e.g.,anti-bodies, e.g., ATG preparation.

[0047] “Thymic irradiation”, as used herein, refers to a treatment inwhich at least 20, and preferably at least 50, 75, 90, or 95% of theadministered irradiation is targeted to the thymus. Whole bodyirradiation, even if the thymus is irradiated in the process ofdelivering the whole body irradiation, is not considered thymicirradiation.

[0048] “MHC antigen”, as used herein, refers to a protein product of oneor more MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes.

[0049] The term “histocompatibility” refers to the similarity of tissuebetween different individuals. The level of histocompatibility describeshow well matched the patient and donor are. The major histocompatibilitydeterminants are the human leukocyte antigens (HLA). HLA typing isperformed between the potential marrow donor and the potentialtransplant recipient to determine how close a HLA match the two are. Thecloser the match the less the donated marrow and the patient's body willreact against each other.

[0050] The term “human leukocyte antigens” or “HLA”, refers to proteins(antigens) found on the surface of white blood cells and other tissuesthat are used to match donor and patient. For instances, a patient andpotential donor may have their white blood cells tested for such HLAantigens as, HLA-A, B and DR. Each individual has two sets of theseantigens, one set inherited from each parent. For this reason, it ismuch more likely for a brother or sister to match the patient than anunrelated individual, and much more likely for persons of the sameracial and ethnic backgrounds to match each other.

[0051] In hematopoietic transplantation, the word “match” relates to howsimilar the HLA typing is between the donor and the recipient. The bestkind of match is an “identical match”. This means that all six of theHLA antigens (2 A antigens, 2 B antigens and 2 DR antigens) are the samebetween the donor and the recipient. This type of match is described asa “6 of 6” match. Donors and recipients who are “mismatched” at oneantigen are considered a “5 of 6” match, and so forth.

[0052] The term “allogeneic donor stem cells” refers to cells fortransplantation in a subject which are derived from a family member(other than an identical twin) or from an unrelated individual, and asused herein includes cells from the same or different species, thelatter being more particularly referred to as “xenogeneic”.

[0053] “Hematopoietic space-creating irradiation”, as used herein,refers to irradiation directed to the hematopoietic tissue, i.e., totissue in which stem cells are found, e.g., the bone marrow. It is ofsufficient intensity to kill or inactivate a substantial number ofhematopoietic cells. It is often given as whole body irradiation.

[0054] “Thymic space” as used herein, is a state created by a treatmentthat facilitates the migration to and/or development in the thymus ofdonor hematopoietic cells of a type which can delete or inactivate hostthymocytes that recognize donor antigens. It is believed that the effectis mediated by elimination of host cells in the thymus.

[0055] “Tolerance”, as used herein, refers to an inhibition of a graftrecipient's immune response which would otherwise occur, e.g., inresponse to the introduction of a nonself MHC antigen into therecipient. Tolerance can involve humoral, cellular, or both humoral andcellular responses. Tolerance, as used herein, refers not only tocomplete immunologic tolerance to an antigen, but to partial immunologictolerance, i.e., a degree of tolerance to an antigen which is greaterthan what would be seen if a method of the invention were not employed.Tolerance, as used herein, refers to a donor antigen-specific inhibitionof the immune system as opposed to the broad spectrum inhibition of theimmune system seen with immunosuppressants. Tolerance is the ability ofthe graft to survive in an MHC mismatched or xenogeneic recipientwithout chronic immunosuppression.

[0056] “Inhibiting immune cell activity” refers to reducing the numberof active immune cells, e.g., thymocytes, T cells, B cells, or NK cells,preferably donor reactive cells, or precursor donor reactive cells, in asubject. Inhibition can include partial inhibition, or partial reduction(as opposed to total elimination) of the number of active immune cells,e.g., T cells.

[0057] The term “relapse” refers to the recurrence of illness afterrecovery; whereas the term “remission” refers to the disappearance ofcancer cells following treatment. Also the period during which thisreduction or disappearance of symptoms occur.

[0058] “Discordant species combination”, as used herein, refers to twospecies in which hyperacute rejection occurs when a graft is graftedfrom one to the other. Generally, discordant species are from differentorders, while non-discordant species are from the same order. Forexample, rats and mice are non-discordant concordant species. Concordantspecies combinations do not exhibit hyperacute rejection. In xenogeneicmethod of the invention, the donor and recipient (subject) can be adiscordant or nondiscordant species combination.

[0059] “Miniature swine”, as used herein, refers to a miniature pigwhich is preferably wholly or partially inbred at at least one MHClocus. The coefficient of inbreeding of the herd which supplies theminiature swine should be at least, 0.70 and more preferably at least0.82. The herd from which donor animals are drawn should be homozygousat the SLA genes.

[0060] (iii) Exemplary Embodiments

[0061] Methods of the invention allow exploitation of theengraftment-promoting and GVL effects of donor T-cells while minimizingGVHD in HLA-mismatched pairs and in xenogeneic methods, allowing manymore patients to benefit from hematopoietic stem cell transplantation.

[0062] GVL effects are mediated by T-cells and other cell types inallogeneic marrow inocula. While GVL effects are often associated withGVHD, these two phenomena can be dissociated. One strategy forseparating these phenomena is the temporal separation of BMT and donorT-cell infusion. Methods of the invention provide initial conditioning,with a mild, relatively non-toxic and non-myeloablative regimen. Thecombination of a mild conditioning regimen and the recovery timepermitted before administration of donor T-cells allows the use of thisapproach in older patients with chronic hematologic malignancies who areotherwise not considered eligible for BMT.

[0063] Because of the high precursor frequency of T lymphocytes reactingto allogeneic MHC molecules, the anti-MHC responses in an allogeneicsetting result in much more potent and more rapid GVL responses than areobserved for MHC-matched BMT. The presence of single HLA antigenmismatches is associated with increased GVL effects in BMT from relateddonors. Because of the very potency of these anti-MHC responses, andbecause of the ubiquity of class I MHC expression, GVHD is a majorimpediment to the full exploitation of this potentially enormous GVLeffect. The greater susceptibility of lymphohematopoietic cells thanother host tissues to destruction by MHC-specific donor T-cells may bedue to the immediate contact of donor cells with host cells within thelymphohematopoietic system. Additional inflammatory stimuli, such ascytokines induced by myeloablative conditioning treatments may berequired to activate endothelial cells and, in combination withactivation-induced increased function of T-cell adhesion molecules,permit T-cell adhesion and migration into GVHD target tissues.Unfortunately, the prior art conditioning of patients for allogeneic BMTparticularly harsh conditioning, may activate inflammatory stimuli, thusexplaining their exquisite sensitivity to the development of GVHD.Methods of the invention avoid GVHD while preserving the stronglymphohematopoietic GVL effects of mismatched allogeneic or xenogeneicdonor tissue without causing GVHD, in part, by the use of conditioningregimens that are less toxic and less pro-inflammatory, followed bydelayed administration of donor T-cells. Such a delay allows recovery ofhost immune resistance to GVHD and/or resolution of theconditioning-induced pro-inflammatory state, and hence decreasedsusceptibility to GVHD. Since the host conditioning used is notmyeloablative, this approach is particularly appropriate for thetreatment of chronic leukemias, for which immediate curativecytoreduction need not be attempted.

[0064] Bone marrow transplantation has not been widely used for thetreatment of chronic lymphocytic leukemia (CLL), an incurable andultimately fatal disease, due largely to the fact that this diseaseoften afflicts older patients who are not considered eligible forallogeneic bone marrow transplantation. Since CLL is a slow-growingleukemia, it is particularly amenable to cure without ablativeconditioning when allogeneic T-cells are administered for their GVLeffect. Additional chronic hematologic malignancies that often afflictolder persons include multiple myeloma, chronic myelogenous leukemia,and low- and intermediate-grade non-Hodgkin's lymphomas are amenable tomethods of the invention.

[0065] Successful allogeneic bone marrow transplantation is oftenlimited by (1) lack of HLA-matched donor (only 25-30% of patients willhave an HLA-phenotypically-identical sibling) and for patients who doundergo an allogeneic BMT, (2) substantial treatment-related mortality,particularly in patents ≧40 years of age, and (3) disease relapse.Methods of the invention expand the availability of transplantation byallowing transplants from HLA-mismatched donors and xenogeneic donors,improve the safety profile of BMT, and enhance thegraft-versus-malignancy effects of mismatched transplantation. Methodsof the invention provide a number of advantages including: (1.) Amoderate dose of cyclophosphamide, a dose of 200 mg/kg, is notmyeloablative and is associated with less regimen-related morbidity andmortality than conventional transplant preparative regimens.Post-chemotherapy hematopoietic recovery is expected in approximately 2weeks following drug administration. While the decreased aggressivenessof the chemotherapy could also mean less tumor cell kill, the decreasedcytoreductive effect of chemotherapy will be outweighed by an enhancedgraft-versus-malignancy effect. (2.) The non-myeloablative conditioningregimen and the presumably lower likelihood of graft-versus-host diseasewill allow for treatment of patients of older age than are consideredfor conventional allogeneic BMT.

[0066] Thus, methods of the invention provide for: less toxicconditioning, which induces less host damage and less pro-inflammatoryresponse to conditioning; partial depletion of donor T cells byadministration to the subject of T cell inhibiting treatment, e.g.,anti-T cell antibodies; and minimization of GVHD by delaying donorleukocyte administration until the pro-inflammatory environment createdby conditioning has receded. Methods of the invention allow the use ofhematopoietic stem cells from mismatched, or xenogeneic, donors, andthus provide increased GVL activity and increases the number ofindividuals who can receive hematopoietic stem cell therapy forhematologic malignancies.

[0067] Methods of the invention also provide for the treatment ofnon-neoplastic disorders or a hemoglobinopathies, e.g., sickle cellanemia, aplastic anemia, thalassemia, or similar disorders.

[0068] In a preferred embodiment, the invention features, a method oftreating a subject e.g., a human, having a hematologic disorder, e.g., ahematologic malignant disorder, e.g., leukemia.

[0069] Certain embodiments of the subject methods also featurepreparative regimens which minimize or eliminate the need formyeloablative treatment, e.g., hematopoietic space-creating irradiation,especially, preparative whole body irradiation.

[0070] One aspect of the present invention provides a method fortreating a subject having a hematologic disorder comprising: (i)administering a myeloreductive nonmyeloablative treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, and (ii) introducing into the subject,allogeneic donor hematopoietic stem cells (donor stem cells) to formchimeric bone marrow in the subject.

[0071] In preferred embodiments each of the recited steps is a separatediscrete administration or agent.

[0072] In methods described herein, the donor can be from the samespecies as the subject, or from a different species. In allogeneicmethods the donor of stem cells and the donor of leukocytes should bethe same individuals. In xenogeneic methods, the subject is a mammal,preferably a primate and more preferably a human. The donor mammal canbe, by way of example, a swine, e.g., a miniature swine, or a nonhumanprimate. In xenogeneic methods the donor of stem cells and the donor ofleukocytes need not be the same individual but can be from differentindividuals which are MHC matched or highly inbred, e.g., inbredminiature swine which are MHC matched.

[0073] While not wishing to be bound by theory, the myeloreductivenon-myeloablative treatment is believed to prepare the subject for theinduction of mixed chimerism and may have a cytoreductive effect oncancer cells. The myeloreductive non-myeloablative treatment should beadministered prior to introduction of the donor hematopoietic stemcells, preferably sufficiently prior to the administration of donorhematopoietic stem cells such that if it includes the administration ofa chemical agent, the chemical agent will be cleared from thecirculatory system, e.g., preferably to a concentration of less than 0.1of the EC₅₀ of the drug for myeloreduction, prior to the administrationof donor hematopoietic stem cells.

[0074] In certain embodiments, the myeloreductive treatment includestreating the subject, prior to introduction of the donor stem cells,with an cytoreductive agent selected from one or more of alkylatingagents (e.g., nitrogen mustards [such as mechloretamine],cyclophosphamide, melphalan and chlorambucil), alkyl sulphonates (e.g.,busulphan), nitrosoureas (e.g., carmustine, lomustine, semustine andstreptozocine), triazenes (e.g., dacarbazine), antimetabolites (e.g.,folic acid analogs such as methotrexate), pyrimidine analogs (e.g.fluorouracil and cytarabine), purine analogs (e.g., fludarabine,idarubicin, cytosine arabinoside, mercaptopurine and thioguanine), vincaalkaloids (e.g., vinblastine, vincristine and vendesine),epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g.,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin andmitomycin), dibromomannitol, deoxyspergualine, dimethyl myleran andthiotepa.

[0075] Preferred myeloreductive non-myeloablative agents are alkylatingagents, e.g., cyclophosphamide, or fludarabine or similar substances,however, hematopoietic space creating antibodies or drugs, e.g.,inhibitors of cell proliferation, e.g., DSG, or an anti-metabolite, e.g.brequinar, or an anti-T cell antibody, e.g., one or both of an anti-CD4or anti-CD8 antibody can be used as a myeloreductive non-myeloablativeagent.

[0076] In preferred embodiments, the myeloreductive non-myeloablativetreatment is sufficiently mild that at lest 10, and more preferably atleast 30, 50, or 75% of the subjects to which it is administered willform mixed chimeras (as opposed to having their bone marrow totallyablated).

[0077] In preferred embodiments, immune cell activity, e.g., T cellactivity, preferably graft reactive T cell activity, is inhibited in thesubject. While not wishing to be bound by theory, the inhibition of Tcells is believed to prepare the subject for the induction of mixedchimerism by inhibition of subject T cell activity which would mount animmune response against the donor hematopoietic stem cells and toinhibit donor T cell activity which would mount an immune responseagainst the subject (GVHD).

[0078] Numerous methods of inhibiting T cell activity are suitable foruse in methods described herein. By way of example, these include:

[0079] the administration of anti-T cell antibodies, e.g., an ATGpreparation, polyclonal or monoclonal antibody directed against CD4,CD8, or CD2 (an anti-CD2 antibody, e.g., the anti-CD2 monoclonalantibody BTI-322 or a humanized version thereof, or an antibody whichoverlaps or binds the epitope recognized by BTI-322, are particularlyuseful);

[0080] the administration of an agent, e.g., an antibody, which blocksor otherwise inhibits a pathway, e.g., a costimulatory pathway, of Tcell activation (agents, e.g., antibodies, which block the CD28-B7pathway, e.g., a CTLA4-IgG fusion protein, or agents, e.g., an antibodywhich blocks the CD40 -gp39 pathway, e.g., an anti-gp39 antibody, areparticularly suited for use in the method), or generally, by theadministration of a treatment which down modulates or otherwise inhibitsone or more of the T cell receptor, CD4 co-receptor, CD8 co-receptor orother receptor or co-receptor which promotes T cell activation ormaturation;

[0081] administration of an IL-12 receptor protein (functionalantagonist, U.S. Ser. No. 5,831,007);

[0082] the administration of substituted dihydrobenzofurans,spirobenzofuran-2(3H)cycloalkanes according to U.S. Ser. No. 5,808,109;

[0083] the administration of anti-asialo antisera;

[0084] the administration of an immunosuppressive agent, e.g., amacrolide, e.g., cyclosporine, FK506, or rapamycin; and

[0085] the administration of thymic irradiation, or other treatmentwhich creates thymic space.

[0086] In certain embodiments, the myeloreductive treatment includestreating the subject with an immunosuppressant regimen, prior tointroduction of the donor stem cells, in an amount sufficient to preventrejection of the donor stem cells by the host immune system. Forexample, such immunosuppressant regimens can include, independently forpre- and post-transplantation is both are carried out, a treatment ofthe subject which inactivates and/or depletes host T-lymphocytes and/ornatural killer (NK) cells in the subject. For example, theimmunosuppressant regimen includes treatment with T cell-depletinganti-CD4 and/or CD8 antibodies, such as anti-thymocyte globulin (ATG),OKT3, LO-CD2a, or Minnesota anti-lymphoblast globulin (MALG).Preferably, the immunosuppressant regimen, both before and aftertransplantation, includes administration of ATG.

[0087] In other embodiments, the immunosuppressant regimen includestreatment with one or more of a macrolide immunosuppressant,azathioprine, steroids (e.g., prednisone, methyl prednisolone), orsub-lethal nonmyleoablative irradiation of lymphocyte-containing tissue.

[0088] Treatments which inhibit T cell activity can be administered atany time in the course of the method but should not be such that donor Tcells will be entirely eliminated. Treatments can be administered priorto, at the same time as, or after, the administration of donorhematopoietic stem cells. Preferably, such treatments are provided bothbefore and after the administration of donor hematopoietic stem cells.Treatment prior to the administration of donor hematopoietic stem cellsis believed desirable in that it will condition the subject for thereceipt of the donor hematopoietic stem cells. Treatment after theadministration of donor hematopoietic stem cells is believed desirablein that it will reduce donor-immune attack on the host and furtherpromote acceptance by the subject of the donor hematopoietic stem cells.

[0089] For best results, treatments to inhibit T cell activity, e.g.,anti-T cell antibodies or cyclosporine, can be administered repeatedly.E.g., such treatment can be administered one, two, three, or more timesprior to donor bone marrow transplantation. Typically, a pre-stem celltreatment, e.g., the administration of antibodies, will be given to thepatient about 1, 2, 3, 4, or 5 days prior to stem cell transplantation.It may be desirable to repeat pre-stem cell administrations every 1-5days until the patient shows excess antibodies in the serum and about80, 90, or 99% depletion of peripheral T cells and then to perform thestem cell transplantation. Treatments can also be administered one, two,three, or more times after donor hematopoietic stem celltransplantation. Typically, a post-stem cell transplant treatment willbe given about 1, 2, 3, 4, or 5 days after bone marrow transplantation.

[0090] In preferred embodiments two or more T cell inhibiting modalitiesor treatments can be combined. In particularly preferred embodiments, anantibody, e.g., an anti-T cell antibody, an immunosuppressive agent,e.g., cyclosporine, and thymic irradiation, are all administered to thesubject. An agent can be administered once, or more than once, but theadministrations should be short term and not chronic or long termadministration. In general, this will mean the treatment is administeredfor not more than 30, 45, 60, 90, or 120 days, and in many treatmentsthis means administration on 1, 2, 3, 4, 5, or fewer days. Cyclosporineand similar agents will generally be administered for not more than 30,45, 60, 90, or 120 days. Antibodies will generally be administered for1, 2, 3, 4, 5, or fewer days.

[0091] While not wishing to be bound by theory, the donor hematopoieticstem cells are believed to provide hematologic function, and to inducetolerance to donor antigen, so as to reduce the subject response to anysubsequent donor tissue, e.g., a donor leukocyte infusion, which isadministered.

[0092] In preferred embodiments, mixed chimerism is induced in thesubject and the state of mixed chimerism is formed in the absence ofhematopoietic space created by space creating irradiation, e.g., wholebody irradiation.

[0093] In preferred embodiments, donor leukocytes are administered tothe subject. While not wishing to be bound by theory, the donorleukocyte administration is believed to provide additional GVLactivity—donor leukocytes are believed to further and very effectivelyreduce the number of cancer cells in the subject. The need for orappropriateness of donor leukocyte administration can be evidenced by alack of increase in donor chimerism, lack of GVHD symptoms, orincomplete tumor regression. Donor leukocyte administration should bedelayed for at least 10, 20, 30, 35 or 60 days after the administrationof any myeloreductive non-myeloablative or other space creatingtreatment. Initial trials showed a delay of about 35 days to besuitable. The donor leukocyte infusion is delayed to avoid introductionof relatively large numbers of donor immune cells into the host duringthe period in which the space creating treatment has inducedpro-inflammatory conditions. Delay allows the host to recover fromconditioning and to be less susceptible to GVHD, especially whenmismatched donor tissue is used. The donor leukocyte infusion convertsthe mixed chimeric state of the subject to one which is fully chimeric,but, the graft cell mediated immune attack will be limited to thehematopoietic compartment, thereby minimizing GVHD and maximizing GVLeffects.

[0094] In preferred embodiments the method includes creating thymicspace in the subject. Thymic space can be created, e.g., by irradiatingthe thymus of the subject, e.g., by administering between 100 and 1,000,more preferably between 300 and 700, e.g., 700 rads, of thymicirradiation, or by administering anti-T cell antibodies in sufficientdose to inactivate thymocytes. Other methods for the creation of thymicspace include: the administration of steroids, corticosteroids,brequinar, or an immune suppressant chemical or drug, e.g., rapamycin,cyclosporin, or FK506. An effective treatment should deplete singlepositive thymocytes to an extent that engraftment and the formation ofmixed chimerism is optimized. In preferred embodiments the subject'ssingle positive thymocytes are depleted by at least 20, 40, 60, or 80%.Treatments which result in between 10 and 90% depletion are preferred.

[0095] In preferred embodiments the subject does not receive additionaltreatments which stimulate the release of a cytokine by mature T cells.E.g., the subject should not receive a substance, e.g., a steroid drug,e.g., Prednisone (17, 21-dihydroxypregna-1, 4diene-3, 11, 20-trione), ata dosage or concentration which stimulates the release of a cytokine bymature T cells in the subject. Preferably, the subject is free of suchtreatment from the time stem cells are first administered until mixedchimerism is established or donor leukocytes administered.

[0096] Preferred embodiments include the administration of an agent,e.g., 15-deoxyspergualin, mycophenolate mofetil, brequinar sodium, or asimilar agent, which inhibits the production, levels, or activity ofantibodies in the subject.

[0097] In preferred embodiments, particularly xenogeneic methods, themethod includes: inhibiting natural killer cells of the subjectpreferably prior to introducing donor tissue into the subject, e.g., byintroducing into the subject an antibody capable of binding to naturalkiller cells of the subject.

[0098] One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. A second anti-mature T cell antibody can be administered aswell, which inhibits T cells as well as NK cells. Anti-T cell antibodiesare present, along with anti-NK antibodies, in anti-thymocyteanti-serum. Repeated doses of anti-NK or anti-T cell antibody may bepreferable. Monoclonal preparations can be used in the methods of theinvention.

[0099] In preferred embodiments, the donor stem cells are provided asallogeneic bone marrow, mobilized peripheral blood cells, or cord bloodcells. The donor stem cells, in some instances, can be expanded ex vivofor transplantation.

[0100] In preferred embodiments, particularly xenogeneic embodiments,the method includes administering donor species stromal cells oradministering donor specific growth factors or cytokines, e.g., SCF orGM-SGF. Where the donor is a miniature swine, the method can includeadministering one or more of swine SCF, swine IL-3, or swine GM-SCF, tothe subject. The method can further include the step of administering afirst or subsequent dose of a cytokine or growth factor to the subject:when the subject begins to show signs of rejection; when the level ofchimerism decreases; when the level of chimerism falls below apredetermined value; when the level of chimerism reaches or falls belowa level where staining with a monoclonal antibody specific for a donorPBMC antigen is equal to or falls below staining with an isotype controlwhich does not bind to PBMC's, e.g. when the donor specific monoclonalstains less than 1-2% of the cells.

[0101] In preferred embodiments, particularly xenogeneic embodiments,the method includes the step of, preferably prior to hematopoietic stemcell transplantation, inhibiting natural subject antibodies, e.g., bydepleting natural antibodies from the blood of the subject. Depletioncan be achieved, by way of example, by contacting the subject's bloodwith an epitope which absorbs preformed anti-donor antibody. The epitopecan be coupled to an insoluble substrate and provided, e.g., as anaffinity column. E.g., an α1-3 galactose linkage epitope-affinitymatrix, e.g., matrix bound linear B type VI carbohydrate, can be used todeplete natural antibodies. Depletion can also be achieved byhemoperfusing an organ, e.g., a liver or a kidney, obtained from amammal of the donor species. (In organ hemoperfusion antibodies in theblood bind to antigens on the cell surfaces of the organ and are thusremoved from the blood.) Other methods for depleting or otherwiseinactivating natural antibodies can be used with the methods describedherein. For example, drugs which deplete or inactivate naturalantibodies, e.g., deoxyspergualin (DSG) (Bristol), or anti-IgMantibodies, can be administered to the recipient of an allograft or axenograft. One or more of, DSG (or similar drugs), anti-IgM antibodies,and hemoperfusion, can be used to deplete or otherwise inactivatesubject natural antibodies in methods of the invention.

[0102] In preferred embodiments: the donor of the hematopoietic stemcell and the donor leukocytes is the same individual. In other preferredembodiments, particularly xenogeneic embodiments, the donor of thehematopoietic stem cell and the donor leukocytes can be differentindividuals, e.g., different individuals which are MHC identical.

[0103] Although methods of the invention generally reduce or eliminatethe need for myeloablative conditioning some embodiments include thestep of, prior to hematopoietic stem cell transplantation, creatinghematopoietic space for the induction of mixed chimerism by irradiatingthe subject with low dose, e.g., less than 400, preferably less than300, more preferably less than 200 or 100 rads, whole body irradiationto partially deplete the bone marrow of the subject. The level of suchtreatment will be very substantially lower than that used in lethalconditioning. As is discussed herein, this treatment can be reduced orentirely eliminated.

[0104] The method can include a further step of treating the subjectwith an immunosuppressant regimen, after introduction of the donor stemcells, in an amount sufficient to prevent a graft-versus-host responsemediated by the donor stem cells.

[0105] Preferably, the pre-transplantation conditioning includesadministration of ATG and cyclophosphamide, and thymic irradiation.Preferably the cyclophosphamide, or other cytoreductive agents, aresubstantially cleared from the patient so as not inhibit proliferationof the transplanted stem cells.

[0106] An important use of the subject method is for allogeneictransplantation of donor stem cells which are mismatched, with respectto the subject, at one or more class II HLA antigens.

[0107] Another important use of the subject method is for allogeneictransplantation of donor stem cells which are mismatched, with respectto the subject, at two or more HLA antigens (either class I or II orboth).

[0108] In several embodiments, particularly where little, and preferablyno GVHD is detected post-transplantation (e.g., at 35 days or longer),the subject method includes the further step of administering allogeneicdonor leukocytes to the subject after introduction of the donor stemcells. The administration of donor leukocytes should be delayedsufficiently from the time of any hematopoietic space creating treatmentsuch that the level of pro-inflammatory cytokines induced by the spacecreating treatment has subsided sufficiently to reduce or substantiallyeliminate GVHD from the donor leukocytes.

[0109] The subject method can also include the management of GVHDresponses post-transplantation by administration of immunosuppressants,or by use of engineered stem cells which give rise to small moleculeablatable T cells or other hematopoietic cells. See, for example, U.S.Pat. No. 5,834,266.

[0110] Thus, in one preferred embodiment, the subject method comprises:(i) identifying a patient having a neoplastic hematopoetic disorder,(ii) administering a myeloreductive treatment to the subject insufficient amount such that mixed hematopoietic chimerism can be inducedin the subject, and (iii) introducing into the subject, allogeneic donorhematopoietic stem cells (donor stem cells) to form stable mixedchimeric bone marrow in the subject.

[0111] In another preferred embodiment, the subject method comprises:(i) identifying a patient having a neoplastic hematopoetic disorder,(ii) administering a myeloreductive treatment to the subject insufficient amount such that mixed hematopoietic chimerism can be inducedin the subject, and (iii) introducing into the subject, allogeneic donorhematopoietic stem cells (donor stem cells) to form mixed chimeric bonemarrow in the subject, wherein the donor stem cells are mismatched, withrespect to the patient, at one or more class II HLA antigens.

[0112] In still another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a myeloreductive treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, and (iii) introducing into the subject,allogeneic donor hematopoietic stem cells (donor stem cells) to formmixed chimeric bone marrow in the subject, wherein the donor stem cellsare mismatched, with respect to the patient, at two or more HLAantigens, e.g., class I and/or class II.

[0113] In yet another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a myeloreductive treatment to the subjectin sufficient amount such that mixed hematopoietic chimerism can beinduced in the subject, (iii) introducing into the subject, allogeneicdonor hematopoietic stem cells (donor stem cells) to form mixed chimericbone marrow in the subject, and (iv) administering apost-transplantation immunosuppression regimen for suppressing ordepleting T-cells in the transplanted donor stem cells.

[0114] In yet another preferred embodiment, the subject methodcomprises: (i) identifying a patient having a neoplastic hematopoeticdisorder, (ii) administering a pre-transplantation conditioning to thesubject in sufficient amount such that mixed hematopoietic chimerism canbe induced in the subject, which pre-transplantation conditioningincludes treating the cells with cyclophosphamide, ATG and thymicirradiation in an amount sufficient to reduce rejection of transplanteddonor stem cells; and (iii) introducing into the subject, allogeneicdonor hematopoietic stem cells (donor stem cells) to form mixed chimericbone marrow in the subject, and (iv) administering ATG to the subjectpost-transplant for suppressing or depleting T-cells in the transplanteddonor stem cells.

[0115] In another embodiment, the subject method includes:

[0116] administering a myeloreductive non-myeloablative treatment, e.g.,an alkylating agent, e.g., cyclophosphamide, or fludarabine or a similarsubstance, to the subject in sufficient amount such that mixedhematopoietic chimerism can be induced in the subject, preferablywithout myeloablative treatment such as whole body irradiation;

[0117] preferably, inhibiting immune cell, e.g., T cell activity, in thesubject;

[0118] introducing into the subject, donor hematopoietic stem cells,preferably mismatched allogeneic or xenogeneic hematopoietic stem cells,e.g., introducing donor bone marrow, to form chimeric bone marrow in thesubject (as is discussed below, if a sufficiently large number of donorhematopoietic stem cells are introduced the myeloreductivenon-myeloablative (hematopoietic space creating) treatment can beminimized or eliminated); and

[0119] optionally, administering to the subject, donor leukocytes,thereby treating the disorder, e.g., relieving or alleviating one ormore symptoms of the disorder. The administration of donor leukocytesshould be delayed sufficiently from the time of any hematopoietic spacecreating treatment such that the level of pro-inflammatory cytokinesinduced by the space creating treatment has subsided sufficiently toreduce or substantially eliminate GVHD from the donor leukocytes.

[0120] In still another embodiment the method of treating a hematologicmalignancy includes the following:

[0121] administering cyclophosphamide to the subject in sufficientamount such that mixed chimerism can be induced in the subject withoutmyeloablative treatment;

[0122] inhibiting T cell activity in the subject by administering thymicirradiation;

[0123] inhibiting T cell activity in the subject by administering ananti-T cell antibody, and a short course of cyclosporine both before andafter the administration of donor hematopoietic stem cells;

[0124] introducing into the subject, allogeneic donor hematopoietic stemcells; and

[0125] optionally, administering to the subject, donor leukocytes.

[0126] In still another embodiment the method is used to treat ahemoglobinopathy, e.g., sickle cell anemia, aplastic anemia,thalassemia, or similar disorder, and includes the following:

[0127] administering a myeloreductive non-myeloablative treatment, e.g.,an alkylating agent, e.g., cyclophosphamide, or fludarabine or a similarsubstance, to the subject in sufficient amount such that mixedhematopoietic chimerism can be induced in the subject, preferablywithout myeloablative treatment such as whole body irradiation;

[0128] preferably, inhibiting immune cell, e.g., T cell activity, in thesubject; and

[0129] introducing into the subject, donor hematopoietic stem cells,e.g., introducing donor bone marrow, to form chimeric bone marrow in thesubject (as is discussed below, if a sufficiently large number of donorhematopoietic stem cells are introduced the myeloreductivenon-myeloablative (hematopoietic space creating) treatment can beminimized or eliminated), to thereby treat the disorder. In thetreatment of non neoplastic disorders, and generally when conversion tofull donor chimerism is not required, the administration of donorleukocytes can be omitted.

[0130] Methods of the invention can be used to treat hematologicdisorders. A hematologic disorder is a disorder in which there is amalfunction in the subject's hematopoietic cells, e.g., thehematopoietic stem cells, which can be treated by replacing orsupplementing the subject's hematopoietic stem cells. Hematologicdisorders include disorders having unwanted cell proliferation, e.g.,hematologic cancers, e.g., hematopoietic and lymphoid malignancies,e.g., leukemia, e.g., chronic lymphocytic leukemia (CLL) and otherchronic hematologic malignancies, including multiple myeloma, chronicmyelogenous leukemia, and low- and intermediate-grade non-Hodgkin'slymphomas. Hematologic disorders also include, non-neoplastic disordersand hemoglobinopathies, e.g., sickle cell anemia, aplastic anemia,thalassemia, and similar disorders.

[0131] As used herein, myeloablative, refers to a treatment in whichdeath, due to marrow failure, in a significant number of recipients,will occur if hematopoietic stem cell transplantation is not given.

[0132] As used herein, non-myeloablative, refers to a treatment whichkills marrow cells but will not, in a significant number of recipients,lead to death from marrow failure.

[0133] As used herein, myeloreductive, refers to a treatment whichcauses cytopenia or anemia.

[0134] Subject, as used herein, refers to a mammal, e.g., a human.

Allogeneic Methods

[0135] The methods described herein can be used where, as between thedonor and recipient, there is any degree of mismatch at MHC loci orother loci which influence graft rejection. Unlike conventional bonemarrow transplantation, mismatch is desirable in methods of theinvention, as mismatch promotes GVL effects. Methods of the inventioncan be used where, as between allogeneic donor and recipient, there is amismatch at at least one MHC locus or at at least one other locus thatmediates recognition and rejection, e.g., a minor antigen locus. Withrespect to class I and class II MHC loci, the donor and recipient canbe: matched at class I and mismatched at class II; mismatched at class Iand matched at class II; mismatched at class I and mismatched at classII; matched at class I, matched at class II. Mismatched, at class I orII, can mean mismatched at one or two haplotypes. Mismatched at MHCclass I means mismatched for one or more MHC class I loci, e.g., in thecase of humans, mismatched at one or more of HLA-A, HLA-B, or HLA-C.Mismatched at MHC class II means mismatched at one or more MHC class IIloci, e.g., in the case of humans, mismatched at one or more of a DP α,a DPβ, a DQ α, a DQ β, a DR α, or a DR β. In any of these combinationsother loci which control recognition and rejection, e.g., minor antigenloci, can be matched or mismatched. It is preferable that there ismismatch at at least one class I or class II locus and, more preferably,mismatch at one class I and one class II locus.

[0136] The methods described herein for inducing tolerance to anallogeneic antigen or allogeneic graft can be used where, as between thedonor and recipient, there is any degree of reactivity in a mixedlymphocyte assay, e.g., wherein there is no, low, intermediate, or highmixed lymphocyte reactivity between the donor and the recipient. Inpreferred embodiments mixed lymphocyte reactivity is used to definemismatch for class II, and the invention includes methods for performingallogeneic grafts between individuals with any degree of mismatch atclass II as defined by a mixed lymphocyte assay. Serological tests canbe used to determine mismatch at class I or II loci and the inventionincludes methods for performing allogeneic grafts between individualswith any degree of mismatch at class I and or II as measured withserological methods. In a preferred embodiment, the invention featuresmethods for performing allogeneic grafts between individuals which, asdetermined by serological and or mixed lymphocyte reactivity assay, aremismatched at both class I and class II.

[0137] In preferred embodiments the donor and the subject are notrelated, e.g., the donor is not a sibling, the offspring of, or theparent of the recipient.

Xenogeneic Methods

[0138] Methods of the invention can use xenogeneic donors. E.g., whenthe subject is a human, the donor can be a non-human primate or a swine,preferably a miniature swine.

Hematopoietic Stem Cells

[0139] Methods of the invention require the introduction of donorhematopoietic stem cells. Administration and engraftment of the donorstem cells converts the subject to a mixed chimera. Because donorhematopoietic stem cells are at a competitive disadvantage to subjecthematopoietic stem cells, it is often desirable to create hematopoieticspace in the donor, in order to promote engraftment of the donor cells.Methods of the invention use mild non-myeloablative methods, e.g., theadministration of cyclophosphamide, to create hematopoietic space.However, if a sufficient number of donor cells are administered, thesubject need not receive space-creating treatment. See e.g., U.S. patentapplication Ser. No. 08/855,705, filed May 8, 1997, hereby incorporatedby reference. Thus, other methods of the invention administer asufficient number of donor hematopoietic stem cells such that thecreation of space, even with mild methods, is not required. Thisapproach is particularly useful in xenogeneic methods, especially thosein which very large numbers of donor hematopoietic stem cells areavailable, e.g., when the donor or donors are inbred miniature swine.

[0140] The number of donor stem cells administered to the recipient canbe increased by either increasing the number of stem cells provided in aparticular administration or by providing repeated administrations ofdonor stem cells.

[0141] Repeated stem cell administration can promote engraftment andmixed chimerism in recipients. In preferred embodiments, particularlyxenogeneic embodiments, multiple administrations of donor stem cells canbe provided. A second (or other subsequent) administration ofhematopoietic stem cell can provided: at least two days, one week, onemonth, or six months after the previous administration of stem cells;when tumor regression is below desired levels; when the level ofchimerism decreases; when the level of chimerism falls below apredetermined value; when the level of chimerism reaches or falls belowa level where staining with a monoclonal antibody specific for a donorPBMC antigen is equal to or falls below staining with an isotype controlwhich does not bind to PBMC's, e.g. when the donor specific monoclonalstains less than 1-2% of the cells; or generally, as is needed tomaintain tumor regression.

[0142] When multiple stem cell administrations are given one or more ofthe administrations can include a number of donor hematopoietic cellswhich is at least twice, is equal to, or is at least 75, 50, or 25% asgreat as, the number of bone marrow cells found in an adult of therecipient species; include a number of donor hematopoietic stem cellswhich is at least twice, is equal to, or is at least 75, 50, or 25% asgreat as, the number of bone marrow hematopoietic stem cells found in anadult of the recipient species. Such large numbers are useful inreducing or eliminating the need for space creating treatment, even mildtreatments.

[0143] The method of introducing stem cells may be altered, particularlyby (1) increasing the time interval between administering hematopoieticstem cells and space creating treatment or leukocyte infusion; (2)increasing the amount of hematopoietic stem cells injected; (3) varyingthe number of hematopoietic stem cell injections; (4) varying the methodof delivery of hematopoietic stem cells; (5) varying the tissue sourceof hematopoietic stem cells, e.g., a fetal liver cell suspension may beused; or (6) varying the donor source of hematopoietic stem cells.Although hematopoietic stem cells derived from the leukocyte donor arepreferable, hematopoietic stem cells may be obtained from otherindividuals or species, or from genetically-engineered inbred donorstrains, or from in vitro cell culture.

Sources of Cells for Allogeneic Stem Cell Transplantation

[0144] A living human donor can provide about 7.5×10⁸ bone marrowcells/kg. Methods of the invention can include the administration of atleast 2 or 3 times this number (per kg) especially when it is desired toreduce or eliminate space creating treatments, and preferably at least10, 15, or 20 times this number. Such large numbers are useful inreducing or eliminating the need for space creating treatment, even mildtreatments. The requisite numbers of bone marrow cells can be providedby the ex vivo expansion or amplification of human stem cells. Ex vivoexpansion is reviewed in Emerson, 1996, Blood 87:3082, herebyincorporated by reference. Methods of ex vivo expansion are described inmore detail in Petzer et al., 1996, Proc. Natl. Acad. Sci. USA 93:1470;Zundstra et al., 1994, BioTechnology 12:909; and WO 95 11692 Davis etal., all of which are hereby incorporated by reference. Sources ofhematopoietic stem cells include bone marrow cells, mobilized peripheralblood cells, and when available cord blood cells.

[0145] The hematopoietic system reconstituting cells administered to therecipient can, in one example, be present in a source population ofbetween 0.2×10⁸ and 4.0×10⁸, or ranges there between, donor bone marrowcells/kg of the recipient's body weight. The bone marrow cells can beobtained from the donor by standard bone marrow aspiration techniquesknown in the art. Bone marrow cells are removed from the donor byplacing a hollow needle into the marrow space and withdrawing a quantityof marrow cells by aspiration.

[0146] Alternatively, the hematopoietic system reconstituting cellsadministered to the recipient can, in one example, be present in asource population of between 1.0×10⁸ and 40×10⁸, or ranges therebetween, donor cytokine mobilized peripheral blood stem cells/kg ofrecipient's body weight. Peripheral blood cells can be obtained from thedonor, for example, by standard phlebotomy or apheresis techniques.Phlebotomy is performed by placing a hollow needle into a vein andwithdrawing a quantity of whole blood using aspiration or gravity.Apheresis is performed in a similar manner to phlebotomy except thewhole blood is anticoagulated and then separated into the constituentformed cellular elements by centrifugation. The mononuclear cellfraction is retained and the remaining plasma and other cellularelements (red blood cells, granulocytes, platelets) are returned to thedonor by intravenous infusion.

[0147] Peripheral blood stem cells can be cytokine mobilized byinjecting the donor with hematopoietic growth factors such asGranulocyte colony stimulating factor (GCSF), granulocyte-monocytecolony stimulating factor (GM-CSF), stem cell factor (SCF)subcutaneously or intravenously in amounts sufficient to cause movementof hematopoietic stem cells from the bone marrow space into theperipheral circulation. The hematopoietic reconstituting cells can alsobe derived from fetal or embryonic human tissue that is processed and/orcultured in vitro so as to increase the numbers or purity of primitivehematopoietic elements.

[0148] In addition, the hematopoietic system reconstituting cellsadministered to the recipient can also be hematopoietic system cellsthat have been enriched from the source population. The sourcepopulation can be either donor bone marrow cells or donor peripheralblood cells. The hematopoietic system reconstituting cells can beenriched from the source population by selecting cells that express theCD34 antigen, using combinations of density centrifugation,immuno-magnetic bead purification, affinity chromatography, andfluorescent activated cell sorting, known to those skilled in the art(Baum et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:2804-8; Lansdorpet al., (1990) J Exp. Med. 172:363-6; Sato et al., (1991) Blood78:967-74; Smith et al., (1991) Blood 77:2122-8; Udomsakdi et al.,(1991) Exp. Hematol 19:338-42; Udomsakdi et al., (1992) Blood80:2513-21.

[0149] The treated mononuclear cells and hematopoietic systemreconstituting cells are typically administered to the recipient in apharmaceutically acceptable carrier by intravenous infusion. Carriersfor these cells can include but are not limited to solutions ofphosphate buffered saline (PBS) containing a mixture of salts inphysiologic concentrations.

Sources of Cells for Xenogeneic Stem Cell Transplantation

[0150] In the case of inbred donor animals, e.g., inbred miniatureswine, very large numbers of stem cells are available, as the numberwhich can be supplied is not limited by the number which can beharvested from a single donor.

[0151] In the case where the recipient is a primate, e.g., a human, andthe donor is a swine, e.g., a miniature swine, 7.5×10⁹ or more, andpreferably, between 7.5×10⁹ and 15×10¹⁰, swine bone marrow cells/kg canbe administered, though this will vary with factors such as theintensity of the preparative regimen and the health of the individualrecipient. Such large numbers are useful in reducing or eliminating theneed for space creating treatment, even mild treatments. As discussedherein, these cells can be provided in more than one administrations.

Treatment of Hematologic Cancers

[0152] The following provides a protocol for the treatment of a humansubject having a hematologic cancer in a preliminary clinical setting.The protocol describes the major components of the treatment, thetherapy to be provided to the patient, pre- and post-treatmentevaluation, and supportive care likely to be needed in the course of thetreatment. This protocol is exemplary of an embodiment of the inventionand is not limiting.

[0153] The treatment consists of four major components:

[0154] 1. Conditioning therapy, e.g., with cyclophosphamide 200 mg/kgand thymic irradiation (7 Gy) and BMT.

[0155] 2. GVHD prophylaxis, e.g., with anti-thymocyte globulin (ATG) andcyclosporine.

[0156] 3. Post-transplant supportive care (antibiotics, transfusionalsupport, hemopoietic growth factors, etc.)

[0157] 4. Donor leukocyte infusions (days +35, +56).

[0158] 5. Thymic irradiation except patients who have received previousmediastinal radiation therapy.

[0159] Scheme of therapy is as follows:

[0160] Day Treatment

[0161] −6 Cyclophosphamide 50 mg/kg

[0162] −5 Cyclophosphamide 50 mg/kg

[0163] −4 Cyclophosphamide 50 mg/kg

[0164] −3 Cyclophosphamide 50 mg/kg

[0165] −2 ATG 15 mg/kg

[0166] −1 Thymic irradiation (7 Gy) ATG 15 mg/kg CYA 5 mg/kg IV

[0167] 0 Bone marrow infusion CYA 5 mg/kg IV

[0168] +1 ATG 15 mg/kg

[0169] +4 CYA 3 mg/kg IV

[0170] +15 CYA 12 mg/kg PO

[0171] +30 CYA 12 mg/kg PO

[0172] +35 Donor leukocyte infusion

[0173] +56 Donor leukocyte infusion Treatment modalities referred to inthe scheme of therapy are as follows:

[0174] A. Cyclophosphamide (Cytoxan™)

[0175] 1. Dosage: cyclophosphamide is administered at a dosage of 50mg/kg on days −6, −5, −4, and −3. cyclophosphamide is dissolved indistilled water and administered over 60 minutes. Dose should becalculated based on actual or ideal body weight, whichever is less.Volume of distilled water to be used is 250 ml for adults.

[0176] 2. Sedation, antinausea: Dexamethasone, Diphenhydramine,Lorazepan and Granisetron prior to cyclophosphamide.

[0177] 3. Because of a 20% incidence of hemorrhagic cystitis, thefollowing plan of fluid administration and management is recommended forprevention:

[0178] a. IV hydration fluids for adults should be at 3000 ml/m²/24hours, beginning 4 hours prior to cyclophosphamide administration.Typically the hydration fluid is D₅NS+20 mEq KCL/liter. This fluidshould be continued for 24 hours after the last dose ofcyclophosphamide.

[0179] b. MESNA at a dose of 15 mg/kg will be administered 15 minutesbefore and 3, 6, and 9 hours after cyclophosphamide (with an additionaldose 24 hours after the fourth IV dose).

[0180] c. Additional KCL and NaHCO₂ may be needed depending on patient'selectrolyte and uric acid status.

[0181] 4. Toxicity and complications

[0182] a. Nausea and vomiting. Variable but usually well controlled withanti emetics.

[0183] b. Uric acid nephropathy. A potential problem that is easilyprevented by high urine flow plus alkalinization and allopurinol.

[0184] c. Fluid retention. cyclophosphamide causes an antidiureticeffect usually counteracted by furosemide administration. Carefulphysical examination and accurate weights three times a day should beable to detect fluid overload early.

[0185] d. Cardiomyopathy. cyclophosphamide causes nonspecific ST changesat this dose level and at total doses ≧200 mg/kg (7.6 grams/m²) fatalcardiac failure due to hemorrhagic necrosis can occur. cyclophosphamideis contraindicated in patients with pre-existing cardiac disease.Patients should receive an EKG on admission, on each daycyclophosphamide is given, and 1 day following cyclophosphamide.

[0186] e. Diarrhea. May be a problem and should be treatedsymptomatically with Tincture of Opium (dose is 1-3 drops/dose) orImmodium. Stool volume loss should be replaced with D₅W and anappropriate electrolyte solution.

[0187] f. Hemorrhagic cystitis. Approximately 50% of patients will havesome hematuria at this dose level, but is usually not symptomatic orsevere unless there is inadequate diuresis. An occasional patient willget severe cystitis despite adequate urine flow.

[0188] g. Alopecia. The patient should be told of hair loss prior todrug administration.

[0189] h. Skin rash. 10-20% of patients develop a diffuse maculopapularrash 24-72 hours following cyclophosphamide. The rash usually resolvesin 24-48 hours.

[0190] i. Anemia. Hematocrit decrements out of proportion to cessationof production will occur at this dose, presumably due to hemolysis.

[0191] j. Electrolyte imbalance. This should be anticipated and dailyelectrolytes followed.

[0192] B. Anti-thymocyte Globulin (ATG; ATGAMTM, Upjohn Co.)

[0193] 1. Dosage: ATG is prepared in 1 liter of normal saline and isgiven at a dose of 15 mg/kg over 10-12 hours on days −2, −1, and +1. Thedose of ATG will be based on ideal or actual body weight, whichever isless.

[0194] 2. Skin Testing

[0195] a. All patients will receive an intradermal skin test (0.1 ml ofa 1 mg/ml solution) and observed for 30 minutes for the presence of awheat/flare reaction. If positive, an alternative treatment plan may beconsidered by the principal investigator. Benadryl 50 mg IV, epinephrine(1:1000 solution) and hydrocortisone 100 mg IV will be available at thebedside in the event of a possible allergic reaction.

[0196] 3. Pre-Medication

[0197] a. All patients will receive dexamethasone 10 mg IV Q 12 hrs ondays −2, −1, and +1. Each ATG infusion will be preceded by Benadryl IVand Tylenol 650 mg.

[0198] 4. Toxicities

[0199] a. Allergic reactions (including anaphylaxis), rash, fevers,rigors, arthralgias, myalgias, dyspnea, serum sickness (including rash,arthritis, proteinuria), hypotension, tachycardia.

[0200] C. Cyclosporine (Sandimmune™, Sandoz Co.)

[0201] 1. Cyclosporine is commercially available and is administeredeither in an intravenous form (mixed in 250 ml of distilled water), oran oral olive oil based solution, or in capsule form (100 and 25 mgcapsules).

[0202] 2. All patients will receive cyclosporine starting on day −1 at adose of 5 mg/kg/day intravenously infused over a period of 20 hoursdaily. The dose will be reduced to 3 mg/kg/day on day +4 until thepatient is able to tolerate p.o. cyclosporine (on or after day +15post-transplant) at a dose of 6 mg/kg twice daily. Cyclosporine will bedose adjusted on the following criteria:

[0203] a. Cyclosporine levels: an attempt will be made to keepcyclosporine dose levels within the therapeutic range (between 250-350mg/ml by monoclonal assay). Given an association between lowcyclosporine levels and the development of acute GVHD, attempts will bemade to keep the level in the high normal range particularly during thefirst 4 weeks post transplant.

[0204] b. Dose reduction should be considered for significant renaldysfunction (e.g. greater than a 50% increase from baseline serumcreatinine level particularly accompanied by a high cyclosporine level).

[0205] c. Careful attention should be given to cyclosporine levels andrenal function in the face of hepatic disease, given the extent ofhepatic metabolism of cyclosporine.

[0206] d. In the absence of acute GVHD, cyclosporine will be tapered anddiscontinued by day +30 post-transplant.

[0207] D. Thymic Irradiation

[0208] 1. 7 Gy of thymic irradiation will be administered in a singledose on day −1.

[0209] 2. Possible toxicities of thymic irradiation include bone marrowsuppression, nausea, vomiting, esophagitis, pneumonitis, pericarditisand secondary malignancy.

[0210] E. Bone Marrow Infusion

[0211] 1. Allogeneic bone marrow will be rapidly infused intravenouslywithout a filter as soon as possible after harvest.

[0212] a. Acute toxicities:

[0213] 1. Pulmonary emboli. Marrow and fat emboli may rarely cause atransient alveolar capillary block and temporary administration of O₂may be necessary.

[0214] 2. Hypotension.

[0215] 3. Volume overload.

[0216] F. Donor Leukocyte Infusion(s)

[0217] 1. Donor peripheral blood mononuclear cells will be collected vialeukophoresis on days +35 and +56 post-transplant. Based on establishedanti-tumor efficacy of 1×10⁷/kg T-cells in CML and reduced risk of GVHDwith this dose compared with ≧5×10⁷/kg T-cells (34), an initial infusion(day +35) of 1×10⁷/kg CD3+ T-cells will be performed. If no GVHD isobserved and fully donor (greater than or equal to 90%) chimerism hasnot been established, or if there is evidence of persistent malignancy,a second dose of 10⁷/kg T-cells will be infused on day +56post-transplant.

[0218] 2. Recipient risks of receiving donor leukocyte infusions includeacute and chronic GVHD and marrow aplasia.

Evaluation

[0219] The following protocol can be used to evaluate prospectiverecipients.

[0220] A. Pre-transplant

[0221] 1. History. A complete history with full details of the patient'sprevious treatment and response will be obtained, including:

[0222] a. Patient exposure to steroids, radiation and antileukemic drugs(total dosage of each antileukemic drug and when given).

[0223] b. Previous or current fever, infections and antibiotictreatment.

[0224] c. Previous CNS involvement and other evidence of extramedullaryleukemia.

[0225] d. Clinical picture at initial presentation including Kamofskyscore.

[0226] e. Prior immunologic and cytogenetic studies of the patient'sleukemic cells.

[0227] 2. Clinical evaluation (all measurements in metric units).

[0228] a. A complete physical examination.

[0229] b. Chest and other radiographs as clinically indicated.

[0230] c. Marrow aspiration and biopsy for staging and cytogenetics.

[0231] d. EKG

[0232] e. Dental consult and evaluation of status of teeth and gums.

[0233] f. Lumbar puncture(s) for determination of presence of CNSleukemia and administration of IT therapy for patients with intermediateto high grade non-Hodgkin's lymphoma.

[0234] g. Pulmonary consult for baseline respiratory studies, with roomair arterial blood gas.

[0235] 3. The following laboratory data should be obtained:

[0236] a. ABO and Rh typing and two-way red cell crossmatch with donor.

[0237] b. HLA typing of patient and available family members andpotential platelet donors.

[0238] c. Hepatitis B surface antigen, HCV, HSV, CMV, HIV and HTLV-1antibody determinations for patient and marrow donors.

[0239] d. Cultures of blood, stool, urine, nose, and throat forpotential pathogenic bacteria, viruses and fungi.

[0240] e. CBC, reticulocyte count, chem 20 and toxoplasma titers. 10 mlserum (2 dry red tops) and 20 cc of heparinized blood (2 green tops) forimmunologic studies to Dr. Spitzer.

[0241] f. Lymphoma or leukemia cells, from marrow or peripheral blood,if available, to be frozen in DMSO for later immunologic studies (sendto Dr. Spitzer).

[0242] The following protocol can be used to monitor patients whoreceive treatment with methods of the invention.

[0243] B. Evaluation during conditioning and the first 100 dayspost-transplantation

[0244] 1. Daily CBC until granulocytes and platelets areself-sustaining; at least three times weekly until discharge, and thenonce or twice weekly until 100 days post-transplant.

[0245] 2. Daily chemistry profile for the first 3 weeks, then asclinically indicated, but at least once weekly.

[0246] 3. Marrow aspiration and biopsy on day 28 and 100post-transplant.

[0247] 4. Chest x-ray every 7 days.

[0248] 5. Serum to serum bank (10 ml clotted blood to Dr. Spitzer) every7 days.

[0249] 6. Viral, bacterial and fungal cultures weekly or when clinicallyindicated or as specified in other protocols until discharge, then asclinically indicated.

[0250] 7. EKG daily during and 1 day after last dose ofcyclophosphamide.

[0251] 8. Daily weights.

[0252] 9. Respiratory function tests per attending.

[0253] 10. Blood for CSP levels every Monday or as per attending (7 mglavender EDTA tube).

[0254] 11. Screening studies for chronic GVHD on day 100 (per Dr.Spitzer).

[0255] 12. Chimerism studies: see study parameter (section XI).

[0256] C. Evaluation following 100 days post-transplant

[0257] 1. Monthly evaluations here for local patients and by referringphysicians for patients who live elsewhere for one year followingengraftment.

[0258] 2. Monthly CBC and chem 20 for 6 months. Marrow aspiration asindicated clinically or required by other protocols.

[0259] 3. Periodic studies as per other specific protocols.

[0260] 4. Complete evaluation every 6 months for 2 years, then annually.

Supportive Care

[0261] The following outlines supportive care which may be appropriate.

[0262] A. Access to vessels. All patients will have placement of asilastic indwelling triple lumen central venous catheter (or have dualdouble lumen catheters) on or before admission.

[0263] B. Hyperalimentation (HAL). Some patients will requirehyperalimentation soon after conditioning. Given the hepatotoxicpotential of HAL, caloric intake will be adjusted to provideapproximately 50 to 75% of the calculated need.

[0264] C. Transfusions.

[0265] 1. Indication. Platelets are transfused to prevent bleeding andan attempt will be made to keep the circulating platelet level >20×10⁹/Lat all times. Packed red blood cells will be transfused to maintain ahematocrit of ≧25%.

[0266] 2. Single donor apheresis platelet products containing a minimumequivalent of 6 units of random donor platelets will be preferentiallyused.

[0267] 3. Patients will have their CMV serostatus determined prior toconditioning therapy. CMV negative patients will receive CMV negativeblood products as available.

[0268] 4. All platelet and red cell transfusions will be white blooddepleted using third generation leukocyte filters.

[0269] 5. Irradiation. All products are irradiated with 2500 rad (from a137/Cs irradiator) prior to infusion to inactivate lymphocytes andprevent a transfusion associated GVHD.

[0270] 6. Problems.

[0271] a. Alloimmunization. Patients frequently become refractory tomismatched platelets due to alloimmunization to HLA and plateletantigens. For practical purposes, this is established by failure todemonstrate an adequate increment 60 minutes after a 4-6 unit equivalenttransfusion. If non-family platelets are required and the patient isimmunized to random donors, attempts should be made to locate anunrelated platelet donor with no HLA antigens in excess of therecipient. Mismatched platelets may be harmful in the presence ofalloimmunization, with chills, fever and a drop in circulating plateletand neutrophil levels.

[0272] b. Consumption. These patients have complicated problems leadingto rapid consumption of platelets, and the distinction between this andalloimmunization is often difficult. Patients who demonstrate anadequate post-transfusion increment but have rapid disappearance ofplatelets are assumed to have consumption and will be given morefrequent platelet transfusions.

[0273] c. Allergic reaction. Chills, fever, and hives occasionally occurdespite adequate circulation of platelets. These are presumed to beallergic reactions to antigens other than HLA or platelet antigens andcan be controlled with diphenhydramine. However, if associated with noincrement or with a decrement, these reactions are probably associatedwith alloimmunization to the transfused platelets and that donor shouldnot be used again.

[0274] D. Management of infections. Principals of infection prophylaxisand treatment will vary according to the spectrum of organisms and theirantibiotic sensitivity and concurrent infection management/antibioticclinical trials. General principals of infection management willinclude:

[0275] 1. reduced bacteria diet

[0276] 2. HEPA filtered or LAF protective isolation

[0277] 3. Oral Ofloxacin 400 mg BID, from admission until ANC is>0.5×10⁹/L for antibacterial prophylaxis.

[0278] 4. Acyclovir 250 mg/m² IV or PO q8 h days −1 until discharge forHSV prophylaxis.

[0279] 5. Fluconazole PO or IV days −1 until ANC is >0.5×10⁹/L forantifungal prophylaxis.

[0280] 6. Broad spectrum antibiotics for fever (T≧1005) in the face ofneutropenia with continuation of antibiotics until ANC is >0.5×10⁹/L.Antibiotic choice will vary but will usually consist of vancomycin and athird generation cephalosporin (e.g. ceftazidime) or imipenem.Aminoglycosides should be avoided if possible in view of potentialsynergistic renal toxicity with CSP and IL2.

[0281] 7. For CMV prophylaxis/therapy:

[0282] a. CMV negative blood products for CMV seronegative recipients.

[0283] b. IVIG 500 mg/kg/week days −8 through +28, then every other weekthrough day +100.

[0284] c. DHPG for positive CMV culture or positive antigen assay.

[0285] These procedures may be employed in combination, as described, orin part These procedures are designed to synergistically prevent theproblem of GVHD while maximizing the GVL effect of donor tissue.

[0286] (iv) Exemplification

[0287] The invention now being generally described will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE 1 Mixed Lymphohematopoietic Chimerism Following aNon-myeloablative Conditioning Regimen and Allogeneic Bone MarrowTransplant (BMT)

[0288] Five patients with chemotherapy (n=5) and radiation (n=2)refractory non-Hodgkin's lymphoma were treated with cyclophosphamide(Cy) 50 mg/kg daily ×4 (days −6 through −3), anti-thymocyte globulin 30mg/kg (days −2, −1, +1) thymic irradiation 700 cGy×1 (n=3) (day −1) andHLA genotypically identical (n=2), phenotypically identical (n=1), or 2antigen mismatched donor BMT (day 0). Intravenous cyclosporine (CYA) wasgiven beginning on day −1 with conversion to p.o. CYA when tolerated.Donor leukocytes (DLI) were given on days +35, (10⁷/kg CD3+ cells) and+56 (5×10⁷/kg CD3+ cells) if no GVHD was present. Median patient age was30 (range 20-45) years. All patients had disease progression duringsalvage chemotherapy or radiation therapy. Toxicities have includedreversible Cy cardiotoxicity (n=1), and capillary leak syndromecoincident with engraftment (n=4). Median times to ANC >0.5 andplatelets >20K have been 16 (range 13-17) and 16 (range 8 to 91+) days.Chimerism analyses of weekly peripheral blood samples and pre-BMT, day+28, and day +100 bone marrow aspirate samples have been performed byvariable number of tandem repeat sequence (VNTR) analysis (HLA matchedBMT) or flow cytometry (HLA mismatched BMT). In recipients ofHLA-matched marrow (n=3) mixed chimerism was seen in all three. Onepatient had conversion to full donor chimerism following DLI anddeveloped grade III GI GVHD. One patient had <10% donor cells; at day+35 he had no detectable donor cells. No donor cells were detectableafter a subsequent DLI. The third patient has 50-70% donor cells on day+28 with grade II cutaneous GVHD. In the two recipients of HLA-2antigenmismatched marrow, >90% donor lymphoid chimerism was seen within 2 weeksof BMT coincident with grade II or III GVHD. In one patient gradualconversion to donor myeloid (neutrophils and monocytes) chimerism wasobserved. In the other patient stable “split” lymphohematopoieticchimerism is present (>90% donor lymphoid, >80% host myeloid). Allpatients are alive at a median of 103 days (range 3y-122 days) followingBMT. Four of five patients are clinically disease-free at the presenttime. Mixed lymphohematopoietic chimerism is achievable following anovel non-myeloablative conditioning regimen and HLA-matched ormismatched BMT. Dramatic anti-tumor responses have been seen in themajority of cases.

EXAMPLE 2 Activation-induced Cell Death in Donor TCR Transgenic T Cellswith Known Host Antigen Specificity

[0289] The expansion and elimination of donor T cell receptor (TCR)transgenic T cells with known host antigen specificity was examined in amurine BMT model. In 2C T-cell receptor transgenic mice (H-2^(b) on B6background), a large fraction of T cells express CD8 and the αβ T-cellreceptor from the cytotoxic T lymphocyte clone 2C, which specificallyrecognizes the MHC class I antigen L^(d) (Sha, W. C. et al., Nature 335,271-274 1988). Lethally irradiated, L^(d+) BALB/c (H-2^(d))mice weretransplanted with 10×10⁶ spleen cells from anti-L^(d) 2C TCR-transgenicB6 mice. By 4 days after BMT, the numbers of 2C CD8 cells in the spleensof BALB/c recipients had increased 14-16 fold over the numberadministered. However, they had drastically decreased to similar numbersto those administered by day 7. By 3-color FCM using 7-AAD(amino-actinomycin-D) for DNA staining, we found that an increasingfraction (4-11%) of GVH-reactive 2C CD8 cells in spleens of 2→BALB/crecipients underwent apoptotic cell death between days 4 and 7 afterBMT, coincident with the observed decrease in absolute numbers of 2C CD8cells in recipients' spleens. In addition, 2C CD8 cells showed reducedexpression of 2C TCR and CD8 on days 4, 7, and 21. They alsodemonstrated anergy upon stimulation with anti-αβTCR and 1B2 (anti-2Cclonotypic mAb) mAbs at these time points. 2C CD8 cells remaining on day+21 expressed the CD45RB^(low) CD44^(high) Mel14^(low), previouslyactivated/memory phenotype. Clinically, the recipients did not show anyevidence of acute GVHD, and all animals survived beyond day 80, althoughsome of them exhibited mild chronic GVHD. The early clonal expansion of2C CD8 cells, downregulation of CD8 and TCR, anergy and deletion of 2CCD8 cells via activation-induced cell death, are all the consequences ofa vigorous immune response. However, this marked monoclonal expansion ofGVH-reactive 2C CD8 cells is incapable of inducing severe acute orsubacute GVHD.

EXAMPLE 3 Mixed Lymphohematopoietic Chimerism and Graft-vs-LymphomaEffects Are Achievable in Adult Recipients Following Non-MyeloablativeTherapy and HLA-Mismatched Donor Bone Marrow Transplantation

[0290] Methods

[0291] Five patients with refractory non-Hodgkins lymphomas underwentBMT from two of six HLA antigen-mismatched (in the GVH direction)donors. Conditioning included pre-transplant cyclophosphamide, pre- andpost-transplant antithymocyte globulin (ATG), and pre-transplant thymicirradiation. Additional GVHD prophylaxis consisted only of cyclosporinA.

[0292] Findings

[0293] Four of four evaluable patients engrafted, and mixedhematopoietic chimerism was established, with donor lymphoidpredominance and varying levels of myeloid chimerism. Two patients arein GVHD-free complete and partial clinical remissions at 460 and 103days post-BMT, respectively.

[0294] Interpretation

[0295] This is the first demonstration that mixed chimerism can beintentionally induced in adult recipients of HLA-mismatched BMT.Moreover, this has been achieved using a non-myeloablative conditioningregimen. The striking anti-lymphoma responses seen in several patientssuggest that allogeneic BMT can have potent immunotherapeutic benefitsin the absence of myeloablative conditioning.

Introduction

[0296] Patients with chemo- and radio-resistant non-Hodgkins lymphomas(NHL) have a very poor prognosis. HLA-identical allogeneic or autologousbone marrow transplantation has led to durable remissions in only 0-23%of patients^(1,2). However, animal studies have shown that MHC-disparatebone marrow transplants can mediate anti-tumor effects that greatlyexceed those achieved with MHC-matched BMT^(3,4). The potential ofHLA-mismatched bone marrow transplantation as immunotherapy forhematologic malignancies has not yet been exploited, largely because ofthe high incidence of intractable GVHD⁵ and of potentially lethalfailure of marrow engraftment associated with standard ablativeconditioning regimens⁶⁻⁸.

[0297] Studies in rodents have shown that mixed hematopoietic chimerasproduced across MHC barriers are resistant to the development of GVHD,even when lymphohematopoietic GVH reactions are intentionally inducedthat convert mixed chimeras to fully allogeneic chimeras ° (M. -G. Wangand M. Sykes, unpublished data). Murine mixed chimeras produced with anon-myeloablative conditioning regimen of T cell-depleting mAbs,cyclophosphamide (CP), and thymic irradiation (TI), can be convertedinto full donor chimeras without developing GVHD when donor lymphocytesare administered 5 weeks post-BMT. We have now adapted the mixedchimerism approach for use in humans with hematologic malignancies,using CP for both cytoreduction of malignancy and as an adjunct to hostimmunosuppression with antithymocyte globulin and TI. We show here thatcytoreductive, immunosuppressive, but non-myeloablative conditioningadministered to patients with refractory hematologic malignancies caninduce stable mixed chimerism with potent graft-versus-lymphoma effects.

Patients and Methods Patients

[0298] The five patients described herein were enrolled at theMassachusetts General Hospital in a trial involving non-myeloablativeconditioning therapy followed by allogeneic BMT, under the auspices ofan MGH Subcommittee for Human Studies-approved protocol. Eligibilitycriteria included chemotherapy-refractory hematologic malignancy, ECOGperformance status of 2 or less, age of 65 years or less and adequateorgan function (as specified by the protocol). A less than three of sixHLA antigen-mismatched related donor was required. Patients and donorswere typed using standard serological techniques for HLA-A and B, andSSOP- or SSP-based analyses for HLADR.

[0299] The characteristics of the HLA-mismatched allogeneic transplantrecipients are listed in Table 1. All five had intermediate- tohigh-grade non-Hodgkins lymphomas that were refractory tochemotherapy±radiotherapy, and received transplants from two of six (A,B, or DR) HLA antigen-mismatched (in the GVH direction) donors.

Conditioning and Transplantation

[0300] Conditioning therapy consisted of intravenous cyclophosphamide(CP) 50 mg/kg/d (with dosing based on actual or ideal body weight,whichever was less) on days −6 through −3, thymic irradiation (700 cGy)on day −1 in patients who had not received previous mediastinalradiation therapy (n=2), and anti-thymocyte globulin (ATG) 30 mg/kg/d(n=2) or 15 mg/kg/d (n=2) on days −2, −1, and +1 (Patients 1 through 4),or 15 mg/kg/d on days −1, +1, +3, +5 (Patient 5). Dexamethasone was usedat a dose of 20 mg/d prior to each dose of CP and at a dose of 10 mgtwice daily with each dose of ATG. Intravenous cyclosporine, 5 mg/kgdaily, was given on day −1 until day +4, when it was reduced to 3mg/kg/d. When oral medication was tolerated, cyclosporine was changed toan oral route at a dose of 6 mg/kg q12 h.

[0301] Donor bone marrow was procured under anesthesia by standardtechniques. A target number of 3×10⁸/kg nucleated cells was sought. Inthe case of minor ABO incompatibility (n=1), plasma was removed fromdonor marrow prior to transplantation. In the case of majorABO-incompatibility (n=1), red blood cells were depleted from the donormarrow using a CS-3000 cell separator (Baxter-Fenwal, Round Lake, Ill.).

Analyses of Chimerism

[0302] Flow cytometry (FCM) was used for analysis of white blood cellsstained with FITC-labeled anti-HLA class I allele-specific mAbs (OneLambda, Inc.; Canoga Park, Calif.) specific for the HLA type of thepatient (HLA-Bw4 or A9) or the donor (HLA-A3). These mAbs were used incombination with PE-, PerCP- or APC-conjugated antibodies to the humanlymphocyte differentiation antigens CD3, CD4, CD8, CD19, and CD56(Becton Dickinson). Staining, flow cytometer calibration and analysiswas performed with standard techniques¹⁰ on approximately 50,000 cellsper analysis tube.

[0303] In four of the five recipients, analysis of minisatellitevariable number of tandem repeats (VNTR) or short tandem repeat (STR)markers 11:12 were also able to distinguish donor and host. Donor orrecipient bands were detectable in mixtures containing as little as 1%of the DNA from the donor or recipient, respectively.

[0304] Erythroid chimerism was determined in Patient 1 (recipient bloodgroup A, donor O) by the gel test method of Lapierre13, using MicroTyping Systems (MTS) (Pompano Beach, Fla.) buffered gel cards withanti-A mAb and anti-H lectin to agglutinate type A and type O RBC,respectively. The sum of A⁺ and H⁺ RBC totaled approximately 100%,indicating that all RBC typed as being of either donor or host origin.

Results Clinical Outcomes

[0305] The clinical courses of the five HLA-mismatched patients in ourtrial are summarized in Table 2. The patient with the longest follow-up(Patient 1) is described in detail. This 20-year-old male presented witha left neck mass in May 1996, followed two months later by rigors andnight sweats. Biopsy of the mass established the diagnosis ofnon-Hodgkins lymphoma, diffuse large cell type. Extensive cervical andmediastinal lymphadenopathy with pulmonary involvement and a pericardialeffusion were present, indicating stage IVB disease. An initial partialresponse to cyclophosphamide, doxorubicin, vincristine and prednisone(CHOP) chemotherapy was followed by progression of cervicallymphadenopathy during the sixth cycle. The patient's cervical diseaseprogressed through subsequent chemotherapy with etoposide, cisplatin,cytosine arabinoside, and methyl-prednisolone (ESHAP) and ifosfamide,carboplatin and etoposide (ICE) and local irradiation (3000 cGy). He wasentered on this protocol, and on May 7, 1997 he underwent allogeneicbone marrow transplantation from his HLA-mismatched brother (2 of 6 HLAantigen mismatch [HLA-A and -B] in the GVH direction and one antigenmismatch [HLA-B] in the HVG direction). In the second weekpost-transplant, an engraftment syndrome (fever and fluid retention) andgrade II acute GVHD (skin and gastrointestinal tract involvement)developed, and responded promptly to corticosteroid therapy. Ameasurable decline in the size of his neck mass was evident immediatelyfollowing chemotherapy. The mass subsequently regressed completely overa period of weeks following transplantation. Restaging at 100 dayspost-transplant confirmed that he was in a partial remission. Steroidswere discontinued approximately six months post-transplant, as thepatient had only minimal cutaneous chronic GVHD. Low dose oralcyclosporine was continued. Approximately seven months post-transplant,an IgG warm antibody-mediated autoimmune hemolytic anemia andthrombocytopenia developed. Though initially responsive to oralcorticosteroids, hemolytic anemia became exacerbated at 9 months,necessitating splenectomy, which showed no evidence of lymphoma uponpathological examination. The thrombocytopenia and hemolytic anemiaresolved, and corticosteroid therapy was tapered. The patient iscurrently in complete remission from his lymphoma 15 months post-BMT,with no evidence of GVHD. Staging evaluations that included CT scans andbone marrow biopsies at seven months and one year post-transplantconfirmed his complete remission status.

[0306] Because of the significant GVHD that developed in Patients 1through 4, Patient 5, who was 51 years old, received a modified protocolthat included less pre-transplant and more post-transplant ATG (15 mg/kgon days −1, +1, +3 and +5). Although grade II GVHD, manifested as fever,skin rash, and elevated liver enzymes, developed in the second week, thepatient responded well to corticosteroids, which have been taperedwithout recurrence of GVHD. Staging at 100 days showed that he is incomplete clinical remission.

Engraftment and Establishment of Mixed Chimerism

[0307] These data are summarized for all five patients in Table 2.Leukopenia and thrombocytopenia occurred within 9 days post-BMT.Leukocyte engraftment (ANC>500/mm3) occurred between 10 and 17 dayspost-BMT in all evaluable patients. Sustained recovery of platelets (to>20,000/mm³) occurred at 9 to 72 days post-transplant in three patients.Patient 2 was still platelet transfusion-dependent at the time of herdeath on day 117. One patient died of pulmonary hemorrhage on day 12,before engrafting.

[0308] Chimerism was assessed by FCM analysis of WBC beginning 8 to 12days post-BMT. All patients showed varying proportions of donor cellsamong lymphocytes, monocytes and neutrophils at the first time pointtested (not shown), and at the time of last follow-up (Table 2). Thetime course for chimerism in these three lineages in Patient 1 ispresented in FIG. 1. Mixed chimerism has been sustained for at least oneyear, when 71% of monocytes, 62% of granulocytes, and 99% of lymphocyteswere donor-derived (FIG. 2). T cells and NK cells were >98%donor-derived, whereas B cells were 33% host-derived and 67%donor-derived. Donor erythrocytes were detectable beginning at 6 to 8weeks post-BMT, and increased to about 80% of RBC by 9 months post-BMT.A marrow aspirate obtained one year post-BMT contained 73% donor and 27%host cells among the non-erythroid elements.

[0309] FCM analyses of Patients 1 through 4 relied on the use of HLAallele-specific mAbs that could specifically identify host, but notdonor cells. The presence of donor cells, for which specific HLAallele-specific mAbs were not available, was verified in Patients 1, 2,and 4 by VNTR or STR analyses, which showed distinct donor bands in eachsample.

Discussion

[0310] Our studies demonstrate that lasting multilineage mixedhematopoietic chimerism, with high levels of donor reconstitution, canbe induced across extensive HLA barriers in adult BMT recipients.Moreover, we show that such chimerism can be achieved in recipientsconditioned with a non-myeloablative regimen, without severe orintractable GVHD, and that it is associated with striking anti-tumorresponses in patients with advanced, refractory non-Hodgkins lymphomas.

[0311] Induction of mixed chimerism across MHC barriers has not, to ourknowledge, been previously reported in adult humans. Lasting mixedchimerism in adults has been achieved across MHC barriers only inrodents^(14:15) Mixed chimerism has been induced in dogs, but only inMHC-identical donor-recipient pairs 6, and transient mixed chimerism hasbeen achieved across MHC barriers in monkeys after non-myeloablativeconditioning¹⁷.

[0312] The lack of myeloablation by the ATG/cyclophosphamide/thymicirradiation conditioning protocol used here is demonstrated by thesurvival of recipient hematopoietic progenitors that are capable ofcontributing to myeloid, lymphoid and erythroid lineages. Furthermore,in a similarly-conditioned recipient of an HLA-matched transplant (datanot shown), secondary failure of donor hematopoiesis was not associatedwith significant cytopenias, due to the ability of surviving hosthematopoietic progenitors to sustain multilineage hematopoiesis.

[0313] Transplantation of HLA-mismatched marrow in myeloablated humanshas been associated with a significant incidence of failure ofengraftment 6, which can be reduced with the use of additionalchemotherapy and immunotherapy^(18;19) Our results suggest that specifictargeting of host immune resistance with the combination ofcyclophosphamide, anti-thymocyte globulin, and thymic irradiation caneffectively overcome host resistance to HLA-mismatched marrowengraftment, despite being less toxic than conventional myeloablativeregimens.

[0314] In several recent studies of non-myeloablative conditioningregimens containing purine analogs in combination with otherchemotherapeutic agents, high levels of donor reconstitution wereachieved in recipients of HLA-matched sibling marrow. Although tumorresponses were obtained primarily in patients with eitherconventional-risk malignancies or with chemosensitive disease in thesestudies, they are consistent with our own data showing that BMT canprovide immunotherapy without host myeloablation²⁰⁻²². However, matchedunrelated donor marrow failed to engraft in two of two patients in oneof these studies²³, suggesting that the host conditioning may be lessimmunosuppressive than that used in our protocol, in which marrowmismatched at one or two of six HLA antigens in the host-vs-graftdirection engrafted in all four cases.

[0315] Despite studies suggesting a graft-versus-lymphoma effect forallogeneic BMT in non-Hodgkins lymphomas, patients with chemoresistantdisease have a very poor prognosis^(2;22;24 27). Striking anti-tumorresponses were achieved in several of our patients, despite the factthat they had very advanced, chemoresistant and even radioresistant,refractory disease. Two of the four evaluable patients are in completeand partial remissions at 460 and 103 days, respectively, and a thirdpatient showed no evidence of disease progression at the time of deathfrom aspergillosis on day 117. Since the only chemotherapy included inour protocol was cyclophosphamide, to which the patients all had priorexposure, the anti-tumor responses seen in our study implicate a potentimmunotherapeutic effect of the donor marrow inoculum. Although longerfollow-up and larger series of patients will be required to determinethe potency and curative potential of this new approach to the treatmentof non-Hodgkins lymphomas, the anti-tumor responses observed suggestthat these may be superior to those achievable with conventional lethalTBI/cyclophosphamide and HLA-matched or closely-matched BMT. This mightoccur because of the more potent alloresponses directed against MHCalloantigens than against minor histocompatibility antigens, which haveled to enhanced GVL effects in rodent studies^(3;4). In addition, thepresence of host-derived professional antigen-presenting cells in mixedchimeras could be associated with enhanced GVL effects for otherreasons, perhaps related to their ability to efficiently present hostalloantigens that are shared by tumor cells.

[0316] The more potent alloresponses generated against MHC disparitiescompared to those against minor histocompatibility antigens usuallyelicits severe GVHD, which has been the major impediment toHLA-mismatched BMT⁵. The patients described here developed GVHD, but inseveral patients it was surprisingly mild and amenable to corticosteroidtherapy. GVHD prophylaxis consisted of cyclosporine plus a singleposttransplant (day +1) treatment with ATG in four patients, and in thefifth patient, whose GVHD was also well-controlled with corticosteroids,an increased proportion of the conditioning ATG was givenpost-transplant rather than pre-transplant. Less severe hostconditioning^(28;29) and the initial presence of host hematopoieticelements³⁰ ³¹ have both been shown to reduce the severity of GVHD inrodents. Larger patient series will determine whether or not our newregimen will allow the routine performance of HLA-mismatched BMT withoutunacceptable GVHD.

References Cites in Example 2

[0317] 1. Ratanatharathon V, Uberti J, Karanes C, et al. Prospectivecomparative trial of autologous versus allogeneic bone marrowtransplantation in patients with non-Hodgkin's lymphoma. Blood 1994;84:1050-1055.

[0318] 2. Jones R J, Ambinder R F, Piantadosi S, Santos G W. Evidence ofa graft-versus-lymphoma effect associated with allogeneic bone marrowtransplantation. Blood 1991; 77:649

[0319] 3. Aizawa S, Sado T. Graft-versus-leukemia effect inMHC-compatible and-incompatible allogeneic bone marrow transplantationof radiation-induced, leukemia-bearing mice. Transplantation 1991; 52:885-889.

[0320] 4. Sykes M, Sachs D H. Genetic analysis of the anti-leukemiceffect of mixed allogeneic bone marrow transplantation. Transplant.Proc. 1989; 21: 3022-3024.

[0321] 5. Clift R A, Storb R. Histoincompatible bone marrow transplantsin humans. Ann. Rev. Immunol. 1987; 5:43-64.

[0322] 6. Anasetti C, Amos D, Beatty P G, et al. Effect of HLAcompatibility on engraftment of bone marrow transplants in patients withleukemia or lymphoma. New Engl. J. Med. 1989; 320:197-204.

[0323] 7. O'Reilly R J, Collins N H, Kernan N, et al. Transplantation ofmarrow depleted of T cells by soybean lectin agglutination and E-rosettedepletion: major histocompatibility complex-related graft resistance inleukemic transplant recipients. Transplant, Proc. 1985; 17:455

[0324] 8. Fleischhauer K, Kernan N A, O'Reilly R J, Dupont B, Yang S Y.Bone marrow-allograft rejection by T lymphocytes recognizing a singleamino acid difference in HLA-B44. New Engl. J. Med. 1990; 323:1818-1822.

[0325] 9. Sykes M, Sheard M A, Sachs D H. Graft-versus-host-relatedimmunosuppression is induced in mixed chimeras by alloresponses againsteither host or donor lymphohematopoietic cells. J. Exp. Med. 1988; 168:2391-2396.

[0326] 10. Preffer F I. Diagnostic cytometry. In: Colvin R B, Bhan A K,McCluskey R T, eds. Diagnostic Immunopathology, 2 ed. New York: RavenPress, 1993: 725-749.

[0327] 11. Schwartz D W M, Glock B, Jungl E M, Mayr W R. Strategy todetect chimerism in allogeneic bone marrow transplant recipients byPCR-amplification fragment length polymorphism analysis ofmicrosatellite polymorphisms. Vox Sang. 1995; 68:139-143.

[0328] 12. Nakao S, Nakasumi T, Chuhjo T, et al. Analysis of late graftfailure after allogeneic bone marrow transplantation: detection ofresidual host cells using amplification of variable number of tandemrepeats. Bone Marrow Transplant. 1992; 9:107-111.

[0329] 13. Lapierre Y, Rigal D, Adam J, et al. The gel test: A new wayto detect red cell antigen-antibody reactions. Transfusion 1990; 30:1091-1113.

[0330] 14. Ildstad S T, Sachs D H. Reconstitution with syngeneic plusallogeneic or xenogeneic bone marrow leads to specific acceptance ofallografts or xenografts. Nature 1984; 307(5947):168-170.

[0331] 15. Sharabi Y, Sachs DbH. Mixed chimerism and permanent specifictransplantation tolerance induced by a non-lethal preparative regimen.J.Exp.Med. 1989; 169: 493-502.

[0332] 16. Storb R, Yi C, Wagner J L, et al. Stable mixed hematopoieticchimerism in DLA-identical littermate dogs given sublethal total bodyirradiation before and pharmacological immunosuppression after marrowtransplantation. Blood 1997; 89:3048-3054.

[0333] 17. Kawai T, Cosimi A B, Colvin R B, et al. Mixed allogeneicchimerism and renal allograft tolerance in cynomologous monkeys.Transplantation 1995; 59:256-262.

[0334] 18. Henslee-Downey P J, Abhyankar S H, Parrish R S, et al. Use ofpartially mismatched related donors extends access to allogeneic marrowtransplant. Blood 1997; 89: 3864-3872.

[0335] 19. Henslee-Downey P J, Parrish R S, Macdonald J S, et al.Combined in vitro and in vivo T lymphocyte depletion for the control ofgraft-versus-host disease following haploidentical marrow transplant.Transplantation 1996; 61:738-745.

[0336] 20. Giralt S, Estey E, Albitar M, et al. Engraftment ofallogeneic hematopoietic progenitor cells with purine analog-containingchemotherapy: Harnessing graft-versus-leukemia without myeloablativetherapy. Blood 1997; 89:4531-4536.

[0337] 21. Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stemcell transplantation and cell therapy as an alternative to conventionalbone marrow transplantation with lethal cytoreduction for the treatmentof malignant and nonmalignant hematologic diseases. Blood 1998;91:756-763.

[0338] 22. Khouri I F, Keating M, Korbling M, et al. Transplant-lite:Induction of graft-versus-malignancy using fludarabine-based nonablativechemotherapy and allogeneic blood progenitor-cell transplantation astreatment for lymphoid malignancies. J.Clin.Oncol. 1998; 16:2817-2824.

[0339] 23. Giralt S, Gajewski J, Khouri I, et al. Induction ofgraft-vs-leukemia (GVL) as primary treatment of chronic myelogenousleukemia. Blood 1997; 90: 418a(Abstract)

[0340] 24. Lundberg J H, Hansen R M, Chitambar C R, et al. Allogeneicbone marrow transplantation for relapsed and refractory lymphoma usinggenotypically HLA-identical and alternative donors. J.Clin.Oncol. 1991;9:1848

[0341] 25. Chopra R, Goldstone A H, Pearce R, et al. Autologous versusallogeneic bone marrow transplantation for non-Hodgkin's lymphoma: Acase-controlled analysis of the European Bone Marrow TransplantationGroup Registry Data. J.Clin.Oncol. 1992; 10:1690

[0342] 26. Ratanatharathorn V, Uberti J, Karanes C, et al. Prospectivecomparative trial of autologous versus allogeneic bone marrowtransplantation in patients with non-Hodgkin's lymphoma. Blood 1994;84:1050

[0343] 27. Verdonck L F, Dekker A W, Lokhorst H M, Petersen E J,Nieuwenhuis H K. Allogeneic versus autologous bone marrowtransplantation for refractory and recurrent low-grade non-Hodgkin'slymphoma. Blood 1997; 90:4201-4205.

[0344] 28. Sprent J, Schaefer M, Gao E, Korngold R. Role of T cellsubsets in lethal graft-versus host disease (GVHD) directed to class Iversus class II H-2differences.I.L3T4+ cells can either augment orretard GVHD elicited by Lyt-2+ cells in Class I-different hosts.J.Exp.Med. 1988; 167:556-569.

[0345] 29. Xun C Q, Thompson J S, Jennings C D, Brown S A, Widmer M B.Effect of total body irradiation, busulfan-cyclophosphamide, orcyclophosphamide conditioning on inflammatory cytokine release anddevelopment of acute and chronic graft-versus-host disease inH-2-incompatible transplanted SCID mice. Blood 1994; 83: 2360-2367.

[0346] 30. Ildstad S T, Wren S M, Bluestone J A, Barbieri S A, StephanyD, Sachs D H. Effect of selective T cell depletion of host and/or donorbone marrow on lymphopoietic repopulation, tolerance, and graft-vs-hostdisease in mixed allogeneic chimeras (B10+B10. D2→B10). J.Immunol. 1986;136:28-33.

[0347] 31. Sykes M, Chester C H, Sachs D H. Protection fromgraft-versus-host disease in fully allogeneic chimeras by prioradministration of T cell-depleted syngeneic bone marrow.Transplantation. 1988; 46:327-330.

[0348] (v) Other Embodiments

[0349] As an alternative or adjunct to hemoperfusion, host antibodiescan be depleted by administration of an excess of hematopoietic cells.

[0350] Stromal tissue can be introduced prior to hematopoietic celltransplant, e.g., BMT. It may be varied by: (1) administering the fetalliver and thymus tissue as a fluid cell suspension; (2) administeringfetal liver or thymus stromal tissue but not both; (3) placing a stromalimplant into other encapsulated, well-vascularized sites, or (4) usingadult thymus or fetal spleen as a source of stromal tissue.

[0351] An anti-CD2 antibody, preferably a monoclonal, e.g., BTI-322, ora monoclonal directed to a similar or overlapping epitope, can be usedin addition to or in place of any anti-T cell antibodies (e.g., ATG) inany method referred to herein.

[0352] Methods of preparing the recipient for transplant ofhematopoietic stem cells may be varied. For instance, recipient mayundergo a splenectomy. The latter would preferably be administered priorto the non-myeloablative regimen, e.g., at day-14.

[0353] (vi) Incorporation by reference

[0354] All of the above-cited references and publications are herebyincorporated by reference.

[0355] (vii) Equivalents

[0356] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents of thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0357] Other embodiments are within the following claims.

What is claimed is:
 1. A method of treating a subject having ahematologic disorder comprising: administering a myeloreductivenon-myeloablative treatment to the subject in sufficient amount suchthat mixed hematopoietic chimerism can be induced in the subject, andintroducing into the subject, allogeneic donor hematopoietic stem cells(donor stem cells) to form chimeric bone marrow in the subject.
 2. Themethod of claim 1 , wherein the myeloreductive treatment includestreating the subject with an immunosuppressant regimen, prior tointroduction of the donor stem cells, in an amount sufficient to preventrejection of the donor stem cells.
 3. The method of claim 1 , comprisingthe further step of treating the subject with an immunosuppressantregimen, after introduction of the donor stem cells, in an amountsufficient to prevent a graft-versus-host response mediated by the donorcells and to prevent rejection of the donor stem cells.
 4. The method ofclaim 2 or 3 , wherein the immunosuppressant regimen includesinactivating or depleting host T-lymphocytes and/or natural killer (NK)cells in the subject.
 5. The method of claim 4 , wherein theimmunosuppressant regimen includes treatment with T cell-depletinganti-CD4 and/or CD8 antibodies.
 6. The method of claim 5 , wherein theimmunosuppressant regimen includes treatment with anti-thymocyteglobulin (ATG).
 7. The method of claim 5 , wherein the immunosuppressantregimen includes treatment with one or more of OKT3, LO-CD2a, Minnesotaanti-lymphoblast globulin (MALG)
 8. The method of claim 2 , wherein theimmunosuppressant regimen includes treatment with thymic irradiation. 9.The method of claim 2 or 3 , wherein the immunosuppressant regimenincludes treatment with sub-lethal nonmyleoablative irradiation oflymphocyte-containing tissue, a costimulatory blocking agent.
 10. Themethod of claim 1 , wherein the myeloreductive treatment furtherincludes treating the subject, prior to introduction of the donor stemcells, with an cytoreductive agent selected from one or more ofalkylating agents, alkyl sulphonates, nitrosoureas, triazenes,antimetabolites, pyrimidine analogs, purine analogs, vinca alkaloids,epipodophyllotoxins, antibiotics, dibromomannitol, deoxyspergualine,dimethyl myleran and thiotepa.
 11. The method of claim 10 , wherein themyeloreductive treatment includes treating the subject withcyclophosphamide.
 12. The method of claim 1 , wherein the donor stemcells are mismatched, with respect to the subject, at one or more classII HLA antigens.
 13. The method of claim 1 , wherein the donor stemcells are mismatched, with respect to the subject, at two or more HLAantigens.
 14. The method of claim 1 , wherein the donor stem cells areprovided as allogeneic bone marrow.
 15. The method of claim 1 , whereinthe donor stem cells are provided as mobilized peripheral blood cells.16. The method of claim 1 , wherein the donor stem cells are provided ascord blood cells.
 17. The method of claim 1 , wherein the donor stemcells are provided as ex vivo expanded stem cells.
 18. The method ofclaim 1 , wherein the donor stem cells are from the same species as thesubject.
 19. The method of claim 1 , wherein the donor stem cells arexenogeneic stem cells from a different species than the subject.
 20. Themethod of claim 1 , wherein the subject is a human.
 21. The method ofclaim 1 , wherein the hematologic disorder includes neoplasticproliferation of hematopoetic cells.
 22. The method of claim 21 ,wherein the hematologic disorder is a leukemia.
 23. The method of claim21 , wherein the hematologic disorder is selected from the groupconsisting of lymphoblastic leukemia, acute or chronic myelogenousleukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myelodysplasticsyndrome, multiple myeloma, and chronic lymphocytic leukemia.
 24. Themethod of claim 21 , wherein the hematologic disorder is refractory tochemotherapy.
 25. The method of claim 24 , wherein the hematologicdisorder is chemorefactory Non-Hodgkin's lymphoma.
 26. The method ofclaim 1 , wherein the hematologic disorder is a non-malignant disorder.27. The method of claim 26 , wherein the hematologic disorder is aninherited erythrocyte abnormalities or inherited immune systemdisorders.
 28. The method of claim 26 , wherein the hematologic disorderis a hemoglobinopathy, e.g., sickle cell anemia, aplastic anemia orthalassemia.
 29. The method of claim 1 , comprising the further step ofadministering allogeneic donor leukocytes to the subject afterintroduction of the donor stem cells.
 30. A method of treating a subjecthaving a hematologic disorder comprising: administering a myeloreductiveand immunosuppressive treatment to the subject in sufficient amount suchthat mixed hematopoietic chimerism can be induced in the subject, andintroducing into the subject, allogeneic donor hematopoietic stem cells(donor stem cells) to form stable mixed chimeric bone marrow in thesubject.
 31. A method of treating a patient having neoplastichematopoetic disorder, comprising: identifying a patient having aneoplastic hematopoetic disorder, administering a myeloreductivenon-myeloablative treatment to the subject in sufficient amount suchthat macroscopic mixed chimerism can be induced in the subject, andintroducing into the subject, allogeneic donor hematopoietic cells(donor stem cells) to form chimeric bone marrow in the subject andinduce a graft-versus-leukemia response and/or graft-versus-lymphomaresponse, which donor stem cells are mismatched, with respect to thepatient, at one or more HLA-A, B or DR antigens.
 32. The use of donorallogeneic hematopoietic cells in the manufacture of a medicament forthe treatment of a hematologic disorder, said medicament administered toa patient conditioned with myeloreductive non-myeloablative treatment,and in an amount sufficient to form chimeric bone marrow in the subject.33. The method of claim 12 , wherein the donor stem cells aremismatched, with respect to the subject, at an HLA-DR antigen.
 34. Themethod of claim 13 , wherein the donor stem cells are mismatched, withrespect to the subject, at two or more HLA-A, B or DR antigens.
 35. Themethod of claim 29 , wherein the allogeneic donor leukocytes areadministered at least 14 days after transplantation.
 36. The method ofclaim 29 , wherein the subject is tested for GVHD, and the allogeneicdonor leukocytes are administered id no GVHD is evident.